Method for production of novel diterpene scaffolds

ABSTRACT

Enzymes and methods are described herein for manufacturing terpenes, including terpenes.

GOVERNMENT FUNDING

This invention was made with government support under 1737898 awarded by the National Science Foundation, and under DE-FC02-07ER64494 and DE-SC0018409 awarded by the U.S. Department of Energy. The government has certain rights in the invention.

This Application claims the benefit of U.S. Provisional Application Ser. No. 62/714,216, filed Aug. 3, 2018, which application is incorporated by reference herein its entirety.

BACKGROUND

Plant-derived terpenoids have a wide range of commercial and industrial uses. Examples of uses for terpenoids include specialty fuels, agrochemicals, fragrances, nutraceuticals and pharmaceuticals. However, currently available methods for petrochemical synthesis, extraction, and purification of terpenoids from the native plant sources have limited economic sustainability.

SUMMARY

Described herein are enzymes useful for production of a variety of terpenes, diterpenes and terpenoids. In some cases, the enzymes synthesize diterpenes. The enzymes were isolated from the mint family (Lamiaceae). Members of the mint family accumulate a wide variety of industrially and medicinally relevant diterpenes. While there are more than 7000 plant species in Lamiaceae, diterpene synthase (diTPS) genes have been characterized from just eleven. The Mint Evolutionary Genomics Consortium, (see website at mints.planthiology.msu.edu) has now sequenced leaf transcriptomes from at least 48 phylogenetically diverse Lamiaceae species, more than doubling the number of mint species for which transcriptomes are available. The available chemotaxonomic and enzyme activity data are described herein for diterpene synthases (diTPSs) in Lamiaceae. The diTPS sequences and terpenes produced are also described herein. One of the new enzymes produces neo-cleroda-4(18),13E-dienyl diphosphate, a molecule with promising applications in agricultural biotechnology as a precursor to potent insect anti-feedants.

Described herein are expression systems that include at least one expression cassette having at least one heterologous promoter operably linked to at least one nucleic acid segment encoding an enzyme with at least 90% sequence identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 57, 59, or 176. In some cases, the expression systems can have more than one expression cassettes or expression vectors, each expression cassette or expression vector can have at least one nucleic acid segment encoding an enzyme with at least 90% sequence identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 57, 59, or 176. Host cells that include such expression systems are also described herein.

Methods are also described herein that include incubating a host cell comprising a heterologous expression system that includes at least one expression cassette having a heterologous promoter operably linked to a nucleic acid segment encoding an enzyme with at least 90% sequence identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 57, 59, or 176. The expression system within host ceil can include more than one expression cassettes or expression vectors.

In addition, methods are described herein for synthesizing a diterpene comprising incubating a terpene precursor with at least one enzyme having at least 90% sequence identity to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 57, 59, or 176. Such methods can include incubating more than one terpene precursor and/or incubating more than one enzyme in a mixture to produce one or more terpenes or terpenoid compounds.

A variety of diterpenes are also described herein.

DESCRIPTION OF THE FIGURES

FIG. 1A-1D illustrate the distribution of diterpenes in Lamiaceae. Note that Table 4 provides a comparison of different sources for data about Lamiaceae diterpene chemotaxonomy. FIG. 1A illustrates diterpene skeletons per genus according to both the Dictionary of Natural Products (DNP) and SISTEMAT. FIG. 1B illustrates the distribution of skeletons among Lamiaceae clades and genera, based on the DNP. Structures are shown for selected skeletons, where black structures are chose where a biosynthetic route is known from Lamiaceae, and gray structures are those for which the pathway remains unknown. FIG. 1C illustrates the distribution of compounds among skeletons, based on the DNP. FIG. 1D illustrates diterpene structures per genus according to both the DNP and the NAPRALERT database. Darker spots indicate overlapping data points, some labels omitted due to space constraints.

FIG. 2A-2B illustrate maximum likelihood trees of diterpene synthase (diTPS) enzymes. FIG. 2A shows a maximum likelihood tree of newly characterized (blue) class II diTPS enzymes. FIG. 2B shows a maximum likelihood tree of newly characterized (blue) class I diTPS enzymes. The maximum likelihood tree of newly characterized (blue) class II and class I diTPS enzymes are shown in the context of previously reported (black) diTPSs from Lamiaceae. The bifunctional ent-kaurene synthase from Physcomitrella patens was used as an outgroup. After each enzyme type are listed the experimentally verified substrates (green) and their products, where the numbers correspond to compound numbers in FIG. 3. Units for scale bars are substitutions per site. Abbreviations for species are listed in Table 5 and those not listed in Table 5 are as follows: Ie, Isodon eriocalyx; Ir, Isodon rubescens; Mv, Marrubium vulgare; Sd, Salvia divinorum; Sm, Salvia miltiorrhiza; Sp, Salvia pomifera; Ss, Salvia sclarea; Vac, Vitex agnus-castus.

FIG. 3A-3B(A)-(H) show structures of products of diterpene synthases from Lamiaceae and a phylogenetic tree was generated from the peptide sequences. FIG. 3A shows products of diterpene synthases from Lamiaceae. Blue numbers indicate compounds experimentally verified to be products of new enzymes identified using the methods described herein. At the center is geranylgeranyl diphosphate (GGPP), a precursor to ail of these diterpenes. The inner ring fire class II products, the product show in the outer ring are class I products derived from the compound in the connected segment of the inner ring. FIG. 3B(A) to 3B(H) show overlapping portions of a phylogenetic tree generated from the peptide sequences from the reference set, alongside those from the new transcriptome data, including established substrates and products for each enzyme.

FIG. 4A-4C illustrate results of activity assays for several enzymes. FIG. 4A shows products detected by gas chromatography from activity assays of Ajuga replans cleroda-4(18),13E-dienyl diphosphate synthase (ArTPS2) and Salvia sclarea sclareol synthase (SsSS) in-vitro with purified protein contacted with GGPP, and in-vivo of N. benthamiana cells that transiently expressed the gene combinations, FIG. 4B shows products detected by gas chromatography from activity assays of PcTPS1+SsSS, in-vitro with purified protein contacted with GGPP, and in-vivo of N. benthamiana cells that transiently expressed the gene combinations. FIG. 4C shows mass spectra for the products of ArTPS2 and PcTPS1, and their combinations with SsSS.

FIG. 5A-5B illustrates the structures that can be produced by the activities of new class I diTPSs. FIG. 5A shows structures that can be generated by the activities of new class 1 diTPSs. Filled in blue boxes indicate which enzymes are capable of each conversion. FIG. 5B illustrates structures that can be produced by the newly characterized enzyme activities including some of the new class II enzymes. Blue genes are newly characterized. Blue square: TPS-e from that position on the key catalyzes the shown transformation. White square: corresponding TPS-e does not catalyze the shown activity. Grey square: corresponding TPS-e was not tested on the substrate.

FIG. 6A-6C illustrate analysis of compounds from O. majorana. FIG. 6A shows GC total ion chromatograms of extracts from N. benthamiana expressing OmTPS1 and OmTPS5, compared to extracts of various tissues of O. majorana. FIG. 6B shows a mass spectrum of peak B, from O. majorana leaf (where peak B is shown in FIG. 6A). FIG. 6C show's a mass spectrum of peak C from O. majorana leaf compared to reference spectrum for palustrinol from the NIST17 library (where peak C is shown in FIG. 6A).

FIG. 7A-7C illustrate the activities of novel Chiococca alba terpene synthases CaTPS1-5. FIG. 7A shows GC-MS-total ion and extracted ion chromatograms illustrating production of ent-kaurene (identified from peak 1) from in vivo assays in N. benthamiana transiently expressing the gene combinations shown. The mass spectrum of peak 1 is shown below the chromatograms, demonstrating that peak 1 is ent-kaurene as identified through direct comparison with biosynthesized authentic standards with reference enzymes. FIG. 7B show's GC-MS-total ion and extracted ion chromatograms illustrating production of ent-dolabradiene (identified from peak 2) from in vivo assays in N. benthamiana transiently expressing the gene combinations shown. The mass spectrum of peak 2 is shown below the chromatograms, demonstrating that peak 2 is ent-dolabradiene as identified through direct comparison with biosynthesized authentic standards with reference enzymes. FIG. 7C shows GC-MS-total ion and extracted ion chromatograms illustrating production of (13R)-ent-manoyl oxide (identified from peak 3) from in vivo assays in N. benthamiana transiently expressing the gene combinations shown. The mass spectrum of peak 3 is shown below the chromatograms, demonstrating that peak 3 is (13R)-ent-manoyl oxide as identified through direct comparison with biosynthesized authentic standards with reference enzymes.

DETAILED DESCRIPTION

Described herein are new enzymes and compounds, as well as methods that fire useful for manufacturing such compounds. The compounds that can be made by the enzymes and methods are new compounds and compounds that were previously difficult to make.

The enzymes described herein are from a variety of mint plant species and can synthesize a variety of terpene skeletons and terpenes.

Terpenes

The enzymes described herein can facilitate synthesis of a variety of terpenes, diterpenes, and terpenoids. For example, the enzymes described herein can facilitate synthesis of terpenes, diterpenes, and terpenoids can generally have the structure of Formula I:

In some cases, the terpenes, diterpenes, and terpenoids can generally have the structure of Formula II:

In some cases, the terpenes, diterpenes, and terpenoids can generally have the structure of Formula III:

The substituents of Formulae I, II, and III can be as follows:

each R₁ can separately be hydrogen or lower alkyl;

R₂ can be hydrogen, lower alkyl, hydroxy, a bond to an adjacent ring carbon, or form a C₄-C₆ cycloheteroalkyl with R₃;

R₃ can be a branched C₅-C₆ alkyl with 0-2 double bonds, can form a C₄-C₆ cycloheteroalkyl with R₂; can form a cycloalkyl with R₄, or can form a cycloheteroalkyl ring with R₄, wherein the C₅-C₆ alkyl can optionally have one hydroxy, phosphate or diphosphate substituent, and wherein each cycloalkyl or cycloheteroalkyl ring can have 0-2 double bonds, and each cycloalkyl or cycloheteroalkyl ring can have 0-2 alkyl or 0-2 alkene substituents;

R₄ can be hydrogen, lower alkyl, lower alkene, hydroxy, a carbon bonded to R₉, an oxygen bonded to R₉, form a cycloalkyl ring with R₃, or form a cycloheteroalkyl ring with R₃, wherein each cycloalkyl ring or cycloheteroalkyl ring can have 0-2 double bonds, and each cycloalkyl ring or cycloheteroalkyl ring can have 0-2 alkyl or 0-2 alkene substituents;

R₅ can be hydrogen, hydroxy, lower alkyl, a lower alkene, a bond with an adjacent carbon, form a cycloalkyl ring with a ring atom of a ring formed by R₃ and R₄, wherein the cycloalkyl ring can have 0-2 double bonds, and the cycloalkyl ring can have 0-2 alkyl or 0-2 alkene substituents;

each R₆ can separately be hydrogen, lower alkyl, lower alkene, or form a bond with an adjacent carbon;

R₇ can be lower alkyl, lower alkene, or form a cycloalkyl ring with a R₅,

R₈ can be lower alkyl, hydroxy, phosphate, diphosphate, or form a bond with an adjacent carbon; or

R₉ can be hydrogen, lower alkyl, lower alkene, ═CH₂, hydroxy, phosphate, diphosphate, form a bond with an adjacent carbon, form a cycloalkyl ring with R₄, or form a cycloheteroalkyl ring with R₄, wherein each cycloalkyl ring or cycloheteroalkyl ring can have 0-2 double bonds, and each cycloalkyl ring or cycloheteroalkyl ring can have 0-2 alkyl or 0-2 alkene substituents.

The alkyl group(s) can have one to ten carbon atoms. In some cases, the alkyl groups can be lower alkyl group(s) (e.g., C1-C6 alkyl groups). In some cases, where substituents such as R₁, R₂, R₅, and R₆ are lower alkyl groups, they can be a C₁-C₃ lower alkyl. In some cases, where substituents such as R₁, R₂, R₅, and R₆ are lower alkyl groups, they are an ethyl or methyl group.

Cycloalkyl groups are cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some cases, the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other cases the number of ring carbon atoms range from 4, 5, 6, or 7. Cycloalkyl groups can include cycloalkyl rings having at least one double bond between 2 carbons (i.e., cycloalkenyl rings). Thus, for example, the A, B and/or C rings can also be a cycloalkenyl group such as a cyclohexenyl, cyclopentenyl, or cyclohexadienyl group. Cycloalkenyl groups can have from 4 to about 8-12 ring members.

Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like. Cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined above. Representative substituted cycloalkyl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4-2,5- or 2,6-disubstituted cyclohexyl groups or mono-, di- or tri-substituted norbornyl or cycloheptyl groups. The term “cycloalkenyl” alone or in combination denotes a cyclic alkenyl group.

Heterocycloalkyl groups include ring groups containing 3 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S. The compounds described herein that have heteroatoms typically have an oxygen heteroatom. In some embodiments, heterocyclyl groups include 3 to about 15 ring members, whereas other such groups have 3 to about 10 ring members. A heterocyclyl group designated as a C₂-heterocyclyl can be a 5-ring with two carbon atoms and three heteroatoms, 6-ring with two carbon atoms and four heteroatoms and so forth. A C₃-heterocyclyl can be a 5-ring with three carbons and two heteroatoms, a 6-ring with three carbons and three heteroatoms, and so forth. A C₄-heterocyclyl can be a 5-ring four carbons and one heteroatom, a 6-ring with four carbons and two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms. A heterocyclyl ring can also include one or more double bonds. The phrase “heterocyclyl group” includes fused ring species including those comprising fused aromatic and non-aromatic groups. For example, a dioxolanyl ring and a benzdioxolanyl ring system (methylenedioxyphenyl ring system) are both heterocyclyl groups within the meaning herein. The phrase also includes polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl. Heterocyclyl groups can be unsubstituted, or they can be substituted. Heterocyclyl groups include, but Eire not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, dihydrobenzofuranyl, indolyl, dihydroindolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Representative substituted heterocyclyl groups can be mono-substituted or substituted more than once, such as, but not limited to, piperidinyl or quinolinyl groups, which are 2-, 3-, 4-, 5-, or 6-substituted, or disubstituted with groups

In some cases, only one of the R₆ groups is a lower alkyl, while the other is hydrogen.

In some cases, R₂ is hydrogen when R₃ forms a ring with R₄.

Although in many diterpenes, each R₆ is a lower alkyl, in some cases one R₆ is a lower alkene white the other is bond that contributes to lower alkene. For example, in some cases the two R₆ groups form a lower alkene together, for example, a ═CH₂ group.

The compounds produced by the enzymes described herein are typically terpenes or diterpenes. Diterpenes are a class of chemical compounds composed of two terpene units, often with the molecular formula. C20H32, though some can include 1-2 heteroatoms or other substituents. Diterpenes generally consist of four isoprene subunits. The positions of various atoms in a diterpene can, for example, be numbered as shown below.

The enzymes described herein can produce compounds with the following skeletons (Sk1-Sk14), where 1-2 of the ring atoms can in some cases be heteroatoms (e.g., oxygen or nitrogen). If a heteroatom is present in it is usually an oxygen atom.

a combination thereof.

Enzymes

The enzymes described herein are from a variety of mint plant species and can synthesize a variety of terpenes, diterpene skeletons, and terpenoid compounds.

For example, an Ajuga reptans miltiradiene synthase (ArTPS3), a Leonotis leonurus sandaracopimaradiene synthase (LITPS4), a Mentha spicata class I diterpene synthase (MsTPS1), an Origanum majorana trans-abienol synthase (OmTPS3), an Origanum majorana manool synthase (OmTPS4), an Origanum majorana palustradiene synthase (OmTPS5), Perovskia atriplicifolia miitiradiene synthase (PaTPS3), Prunella vulgaris miltiradiene synthase (PvTPS1), Salvia officinalis miitiradiene synthase (SoTPS1) were identified and isolated as described herein.

Eight of these enzymes, ArTPS3, LITPS4, MsTPS1, OmTPS4, OmTPS5, PaTPS3, PvTPS1, and SoTPS1 can convert a labda-13-en-8-ol diphosphate ((+)-8-LPP) [compound 10]) to 13R-(+)-manoyl oxide [8].

The ArTPS3, LITPS4, OmTPS4, OmTPS5, PaTPS3, PvTPS1, and SoYPS1 enzymes can also convert peregrinol diphosphate (PgPP) [5] to a combination of compounds 1, 2, and 3, as illustrated below.

However, MsTPS1 produced only compound 3 from compound 5, white the OmTPS3 enzyme produced only 1, and 2. The OmTPS4 enzyme produced compound 4 (shown below) in addition to compounds 1, 2, and 3.

The ArTPS3, PaTPS3, PvTPS1, and SoTPS1 enzymes can also convert (+)-copalyl diphosphate ((+)-CPP) [31]) to miltiradiene [32],

However, LITPS4 and MsTPS1 converted (+)-copalyl diphosphate ((+)-CPP) [31]) to sadaracopimaradiene [27], white OmTPS3 converted (+)-copalyl diphosphate ((+)-CPP) [31]) to trans-biformene [34],

The Ajuga reptans miltiradiene synthase (ArTPS3) has the amino acid sequence shown below (SEQ ID NOT).

1 MSLSFTIKVT PFSGQRVHSS TESFPIQQFP TITTKSAMAV 41 KCSSLSTATV SFQDFVGKIR DTINGKVDNS PAATTIHPAD 81 IPSNLCVVDT LQRLGVDRYF QSEIDSVLND TYRFWQQKGE 121 DIFTDVACRA MAFRLLRVKG YEVSSDELAS YAEQEHVNLQ 161 PSDITTVIEL YRASQTRLYE DEGNLEKLHT WTSNFLKQQL 201 QSETISDEKL HKQVEYYLKN YHGILDRACV RQSLDLYDIN 241 QYQNLKSTDR FPTLSNEDLL EFAKQDFNFC QAQHQKELQQ 281 LQRWYADCKL DTLTYGRDVV RVASFLTAAI FCEPEFSDAR 321 LAFAKHIILV TRIDDFFDHG GSIEESYKIL DLVKEWEDKP 361 AEEYPSKEVE ILFTAVYNTV NDLAEMAYIE QGRSIKPLLI 401 KLWVEILTSF KKELDSWTED TELTLEEYLA SSWVSIGCRI 441 CSLNSLQFLG ITLSEEMLSS EECMELCRHV SSVDRLLNDV 481 QTFEKERLEN TINSVSLQLA EAQREGRTIT EEEAMSKIKD 521 LADYHRRQLM QMVYKDGTIF PRQCKDVFLR VCRIGYYLYA 561 SGDEFTTPQQ MMGDMKSLVY EPLNTSSS A nucleic acid encoding the Ajuga reptans miltiradiene synthase (ArTPS3) with SEQ ID NOT is shown below as SEQ ID NO:2.

1 ATGTCACTCT CGTTCACCAT CAAAGTCACC CCCTTTTCGG 41 GCCAGAGAGT TCACAGCAGC ACAGAAAGCT TTCCAATCCA 81 ACAATTTCCA ACGATCACCA CCAAATCCGC CATGGCTGTC 121 AAATGCAGCA GCCTCAGTAC CGCAACAGTA AGCTTCCAGG 161 ATTTCGTCGG AAAAATCAGA GATACGATCA ACGGGAAAGT 201 TGACAATTCT CCAGCAGCGA CCACTATTCA TCCTGCAGAT 241 ATACCCTCCA ATCTCTGCGT GGTGGATACC CTCCAAAGAT 281 TGGGAGTTGA CCGTTACTTC CAATCTGAAA TCGACAGCGT 321 TCTTAACGAC ACATACAGGT TCTGGCAGCA GAAAGGAGAA 361 GATATCTTCA CTGATGTTGC TTGTCGTGCA ATGGCATTTC 401 GACTTTTGCG AGTTAAAGGA TATGAAGTTT CATCAGATGA 521 ACTCGCTTCG TATGCTGAAC AAGAGCATGT TAACCTGCAA 561 CCAAGTGACA TAACTACGGT TATCGAGCTT TACAGAGCAT 601 CACAGACAAG ATTATATGAA GACGAGGGCA ATCTTGAGAA 641 GTTACATACT TGGACTAGCA ATTTTCTGAA GCAACAATTG 681 CAGAGTGAAA CTATTTCTGA CGAGAAATTG CACAAACAGG 721 TGGAGTATTA CTTGAAGAAC TACCACGGCA TACTAGACCG 761 TGCTGGAGTT AGACAAAGTC TCGATTTATA TGACATAAAC 801 CAATACCAGA ATCTAAAATC TACAGATAGA TTCCCTACTT 841 TAAGTAACGA AGATTTACTT GAATTCGCGA AGCAAGATTT 881 TAACTTTTGC CAAGCTCAAC ACCAGAAAGA GCTTCAGCAA 921 CTGCAAAGGT GGTATGCGGA TTGTAAATTG GATACATTGA 961 CTTACGGAAG AGATGTGGTA CGTGTTGCAA GTTTCCTGAC 1001 AGCTGCAATT TTTGGTGAGC CTGAATTCTC TGATGCTCGT 1041 CTAGCCTTCG CCAAACACAT CATCCTCGTG ACACGTATTG 1081 ATGATTTCTT CGATCATGGT GGGTCTATAG AAGAGTCATA 1121 CAAGATCCTG GATTTAGTAA AAGAATGGGA AGATAAGCCA 1161 GCTGAGGAAT ATCCTTCCAA GGAAGTTGAA ATCCTCTTTA 1201 CAGCAGTATA TAATACAGTA AATGACTTGG CAGAAATGGC 1241 TTATATTGAG CAAGGCCGTT CCATTAAACC TCTTCTAATT 1281 AAACTGTGGG TTGAAATACT GACAAGTTTC AAGAAAGAAC 1321 TGGATTCATG GACAGAAGAC ACAGAACTAA CCTTGGAGGA 1361 GTACTTGGCT TCCTCCTGGG TGTCGATCGG TTGCAGAATC 1401 TGCAGTCTCA ATTCGCTGCA GTTCCTTGGT ATAACATTAT 1441 CCGAAGAAAT GCTTTCAAGC GAAGAGTGCA TGGAGTTGTG 1481 TAGGCATGTT TCTTCAGTCG ACAGGCTACT CAATGACGTG 1521 CAAACTTTCG AGAAGGAACG CCTAGAAAAT ACGATAAACA 1561 GTGTGAGCCT ACAGCTAGCA GAAGCTCAGA GAGAAGGAAG 1601 AACCATTACA GAAGAGGAGG CTATGTCAAA GATTAAAGAC 1641 CTGGCTGATT ATCACAGGAG ACAACTGATG CAGATGGTTT 1681 ATAAGGATGG GACCATATTT CCGAGACAAT GCAAAGATGT 1721 CTTTTTGAGG GTATGCAGGA TTGGCTACTA CTTATACGCG 1761 AGCGGCGATG AATTCACTAC TCCACAACAA ATGATGGGGG 1801 ATATGAAATC ATTGGTTTAT GAACCCCTAA ACACTTCATC 1841 CTCTTGA

The Leonotis leonurus sandaracopimaradiene synthase (LITPS4) has the amino acid sequence shown below (SEQ ID NO:3).

1 MSVAFNLIVV RFPGHGIQSS RETFPAKIIT RTKSSMRFQS 41 SLNTSTDFVG KIREMIRGKT DNSINPLDIP STLCVIDTLH 81 SFGIDRYFQS EINSVLHHTY RLWNDRNNII FKDVICCAIA 121 FRLLRVKGYQ VSSDELAPFA QQQVTGLQTS DIATILELYR 161 ASQERLHEDD DTLDKLHDWS SNLLKLHLLN ENIPDHKLHK 201 RVGYFLKNYH GMLDRVAVRR NIDLHNINHY QIPEVADRFP 241 TEAFLEFSRQ DFNICQAQHQ KELQQLHRWY ADCRLDTLNH 281 GTDVVHFANF LTSAIFGEPE FSEARLAFAK QVILITRMDD 321 FFDHDGSREE SHKILHLVQQ WKEKPAEEYG SKEVEILFTA 361 VYTTVNSLAE KACMEQGRSV KQLLIKLWVE LLTSFKKELD 401 SWTEKMALTL DEYLSFSWVS IGCRLCILNS LQFLGIKLSE 441 EMLWSQECLD LCRHVSSVVR LLNDLQTFKK ERIENTINGV 481 DVQLAARKGE RAITEEEAMS KIKEMADHHR RKLMQIVYKE 521 GTIFPRECKD VFLRVCRIGY YLYSGDELTS PQQMKEDMKA 561 LVHESSS A nucleic acid encoding the Leonotis leonurus sandaracopimaradiene synthase (LITPS4) with SEQ ID NO:3 is shown below as SEQ ID NO:4.

1 ATGTCGGTGG CGTTCAACCT CATAGTCGTC CGTTTTCCGG 41 GCCATGGAAT TCAGAGCAGT AGAGAAACTT TTCCAGCCAA 81 AATTATTACC AGAACTAAAT CAAGCATGAG ATTCCAAAGC 121 AGCCTCAACA CTTCAACAGA TTTCGTGGGA AAAATAAGAG 161 AGATGATCAG AGGGAAAACT GATAATTCTA TTAATCCCCT 201 GGATATTCCC TCCACTCTAT GCGTAATCGA CACCCTACAC 241 AGCTTCGGAA TTGATCGCTA CTTTCAATCC GAAATCAACT 281 CTGTTCTTCA CCACACATAC AGATTATGGA ACGACAGAAA 321 TAATATCATC TTCAAAGATG TCATTTGCTG CGCAATTGCC 361 TTTAGACTTT TGCGAGTGAA AGGATATCAA GTCTCATCAG 401 ATGAACTGGC GCCATTTGCC CAACAACAGG TGACTGGACT 441 ACAAACAAGC GACATTGCCA CGATTCTAGA GCTCTACAGA 481 GCATCACAGG AGAGATTACA CGAAGACGAC GACACTCTTG 521 ACAAACTACA TGATTGGAGC AGCAACCTTC TGAAGCTGCA 561 TCTGCTGAAT GAGAACATTC CTGATCATAA ACTGCACAAA 601 CGGGTGGGGT ATTTCTTGAA GAACTACCAT GGCATGCTAG 641 ATCGCGTTGC GGTTAGACGA AACATCGACC TTCACAACAT 681 AAACCATTAC CAAATCCCAG AAGTTGCAGA TAGGTTCCCT 721 ACTGAAGCTT TTCTTGAATT TTCAAGGCAA GATTTTAATA 761 TTTGCCAAGC TCAACACCAG AAAGAACTTC AGCAACTGCA 801 TAGGTGGTAT GCAGATTGTA GATTGGACAC ACTGAATCAC 841 GGAACAGACG TAGTACATTT TGCTAATTTT CTAACTTCAG 881 CAATITTCGG AGAGCCTGAA TTCTCCGAGG CTCGTCTAGC 921 CTTTGCTAAA CAGGTTATCC TAATAACACG TATGGATGAT 961 TTCTTCGATC ACGATGGGTC TAGAGAAGAA TCACACAAGA 1001 TCCTCCATCT AGTTCAACAA TGGAAAGAGA AGCCCGCCGA 1041 AGAATATGGT TCAAAGGAAG TTGAGATCCT CTTTACAGCA 1081 GTGTACACTA CAGTAAATAG CTTGGCAGAA AAGGCTTGTA 1121 TGGAGCAAGG CCGTAGTGTC AAACAACTTC TAATTAAGCT 1161 GTGGGTCGAG CTGCTAACAA GTTTCAAGAA AGAATTGGAT 1201 TCATGGACGG AGAAGATGGC GCTAACCTTG GATGAGTACT 1241 TGTCTTTCTC CTGGGTGTCA ATTGGCTGCA GACTCTGCAT 1281 TCTCAATTCC CTGCAATTTC TTGGGATAAA ATTATCTGAA 1321 GAAATGCTGT GGAGTCAAGA GTGTCTGGAT TTATGCCGGC 1361 ATGTTTCATC AGTGGTTCGC CTGCTCAACG ATTTACAAAC 1401 TTTCAAGAAG GAGCGCATAG AAAATACGAT AAACGGTGTG 1441 GACGTTCAGC TAGCTGCTCG TAAAGGCGAA AGAGCCATTA 1481 CAGAAGAGGA GGCCATGTCC AAGATTAAGG AAATGGCTGA 1521 CCATCACAGG AGAAAACTGA TGCAAATTGT GTATAAAGAA 1561 GGAACCATTT TTCCAAGAGA ATGCAAAGAT GTGTTTTTGA 1601 GAGTGTGCAG GATTGGCTAC TATCTCTACT CGGGCGATGA 1641 GTTAACTTCT CCACAACAAA TGAAGGAGGA TATGAAAGCG 1681 TTGGTACATG AATCATCCTC TTGA

The Mentha spicata class I diterpene synthase (MsTPS1) has the amino acid sequence shown below (SEQ ID NO:5).

1 MSSIRNLSLH IDLPKAEKKL VEKIRERIRN GRVEMSPSAY 41 DTAWVAMVPS RGYSGRPGFP ECVDWIIENQ NPDGSWGLDS 81 DQPLLVKDSL SSTLACLLAL RKWKTHNQLV QRGMEFIDSR 121 GWAATDDDNQ ISPIGFNIAF PAMINYAKEL NLTLPLHPPS 161 IHSLLHIRDS EIRKRNWEYV AEGVVDDTSN WKQIIGTHQR 201 NNGSLFNSPA TTAAAVIHSH DDKCFRYLIS TLENSNGGWV 241 PTIYPYDIYA PLCMIDTLER LGIHTYFEVE LSGIFDDIYR 281 NWQEREEEIF CNVMCRALAF RLLRMRGYHV SSDELAEFVD 321 KEEFFNSVSM QESGEGTVLE LYRASLTKIN EEERILDKIH 361 AWTKPFLKHQ LLNRSIRDKR LEKQVEYDLK NFYGALVRFQ 401 NRRTIDSYDA KSIQTSKTAY RCSTVYNEDF IHLSVEDFKI 441 SRAQYLKELE EMNKWYSDCR LDLLTKGRNA CRESYILTAA 481 IIVDPHESMA RISYAQSILL ITVFDDFFDH YGSKEEALNI 521 IDLVKEWKPA GSYCSKEVEI LFTALHDTIN EIAAKADAEQ 561 GFSSKQQLIN MWVELLESAV REKDSLSXNK VSTLEEYLSF 601 APITIGCKLC VLTSVHFLGI KLSEEIWTSE ELSSLCRHGN 641 VVCRLLNDLK TYEREREENT LNSVSVQTVG GGVSEEEAVT 681 KVEEVLEFHR RKVMQLACRR GGSSVPRECK ELVWKTCTIG 721 YCLYGHDGGD ELSSPKDILK DINAMMFEPL K A nucleic acid encoding the Mentha spicata class I diterpene synthase (MsTPS1) with SEQ ID NO:5 is shown below as SEQ ID NO:6.

1 ATGAGTTCCA TTCGAAATTT AAGTTTGCAT ATTGATCTGC 41 CAAAGGCCGA GAAGAAGTTG GTTGAGAAAA TCAGAGAGAG 81 GATAAGAAAT GGGAGGGTGG AGATGTCGCC GTCGGCTTAC 121 GACACCGCGT GGGTGGCCAT GGTGCCGTCT CGAGGATATT 161 CCGGCAGGCC GGGTTTCCCG GAGTGCGTGG ATTGGATAAT 201 CGAGAACCAG AATCCCGACG CGTCGTGGGG TTTGGATTCG 241 GATCAACCAC TTCTGGTCAA AGACTCCCTC TCGTCCACCT 281 TGGCATGCCT ACTTGCCCTG CGTAAATGGA AAACACACAA 321 CCAACTAGTG CAAAGGGGCA TGGAGTTCAT CGACTCCCGT 361 GGTTGGGCTG CAACTGATGA TGACAATCAG ATTTCTCCTA 401 TTGGATTCAA TATTGCCTTT CCTGCAATGA TTAATTACGC 441 CAAAGAGCTT AATTTAACTC TGCCTCTACA TCCACCTTCG 481 ATTCATTCAT TGTTACACAT TAGAGATTCA GAAATAAGAA 521 AGCGAAACTG GGAATACGTA GCTGAAGGAG TAGTCGACGA 561 TACAAGCAAT TGGAAGCAAA TAATCGGCAC GCATCAAAGA 601 AATAATGGAT CCTTGTTCAA CTCACCTGCT ACCACTGCAG 641 CTGCTGTTAT TCACTCTCAC GACGATAAAT GTTTCCGATA 681 TITGATCTCC ACTCTTGAGA ATTCTAACGG TGGATGGGTA 721 CCAACTATCT ATCCATACGA TATATACGCT CCTCTCTGCA 761 TGATCGATAC GCTAGAAAGA TTAGGAATAC ACACATATTT 801 TGAAGTTGAA CTCACCGGCA TTTTTGATGA CATATACAGG 841 AATTGGCAAG AGAGAGAAGA AGAGATCTTT TGTAATGTTA 881 TGTGTCGACC TCTGGCATTT CGGCTTCTAC GAATGAGGGG 921 ATATCATGTT TCATCTGATG AACTAGCAGA ATTTGTGGAC 961 AAGGAGGAGT TTTTTAATAG CGTGAGCATG CAAGAGAGCG 1001 GCGAAGGCAC AGTGCTTGAG CTTTACAGAG CTTCACTCAC 1041 AAAAATCAAC GAAGAAGAAA GGATTCTCGA CAAAATTCAT 1081 GCATGGACCA AACCATTTCT CAAGCACCAG CTTCTCAACC 1121 GCAGCATTCG CGACAAACGA TTAGAGAAGC AGGTGGAATA 1161 CGACTTGAAG AACTTCTACG GCGCACTAGT CCGATTCCAG 1201 AACAGAAGAA CCATCGACTC ATACGATGCT AAATCAATCC 1241 AAATTTCGAA AACAGCATAT AGGTGCTCTA CAGTTTACAA 1281 TGAAGACTTC ATCCATTTAT CCGTTGAGGA CTTCAAAATC 1321 TCCCGAGCAC AATACCTAAA AGAACTTGAA GAAATGAACA 1361 AGTGGTACTC TGATTGTAGG TTGGACCTCT TAACTAAAGG 1401 AAGAAATGCA TGTCGAGAAT CTTACATTTT AACAGCTGCA 1441 ATCATTGTCG ATCCTCACGA ATCCATGGCT CGLATCTCTT 1481 ACGCTCAATC TATTCTTCTT ATAACTGTTT TCGACGACTT 1521 TTTCGATCAT TATGGGTCTA AAGAAGAGGC TCTCAATATT 1561 ATTGATCTAG TCAAGGAATG GAAGCCAGCT GGCAGTTACT 1601 GCTCCAAAGA AGTGGAGATT TTGTTTACTG CATTACACGA 1641 CACGATAAAT GAGATTGCAG CCAAGGCTGA TGCAGAGCAA 1681 GGCTTTTCTT CCAAACAACA GCTTATCAAC ATGTGGGTGG 1721 AGCTACTTGA GAGCGCCGTG AGAGAAAAGG ACTCGCTGAG 1761 TGGNAACAAA GTGTCGACTC TAGAAGAGTA CTTATCTTTC 1801 GCACCAATCA CCATCGGCTG CAAACTTTGC GTCCTGACGT 1841 CTGTCCATTT CCTCGGAATC AAACTGTCCG AGGAAATCTG 1881 GACTTCCGAG GAGTTGAGCA GTCTGTGCAG GCACGGCAAT 1921 GTTGTCTGCA GACTGCTCAA CGACCTCAAG ACTTACGAGA 1961 GAGAGCGCGA AGAGAACACG CTCAACAGCG TGAGCGTGCA 2001 GACAGTGGGA GGAGGCGTTT CGGAGGAAGA GGCGGTGACG 2041 AAGGTGGAGG AGGTGTTGGA ATTTCATAGA AGAAAAGTGA 2081 TGCAGCTCGC GTGTCGAAGA GGAGGAAGCA GTGTTCCGAG 2121 AGAATGTAAG GAGCTGGTGT GGAAGACGTG CACGATAGGT 2161 TACTGCTTGT ACGGTCACGA CGGAGGCGAT GAGTTATCGT 2201 CTCCGAAGGA TATTCTAAAG GACATTAATG CAATGATGTT 2241 TGAGCCTCTC AAGTGA

A Nepeta mussinii ent-kaurene synthase (NmTPS2) was identified and isolated as described herein. This NmTPS2 enzyme was identified as an ent-kaurene synthase, which converts ent-CPP [16] into ent-kaurene [19].

The Nepeta mussinii ent-kaurene synthase (NmTPS2) has the amino acid sequence shown below (SEQ ID NO:7).

1 MSLPLSSCVL FPPNDSRFPV SRFSRASASL EVGLQGATSA 41 KVSSQSSCFE ETKRRITKLF HKDELSVSTY DTAWVAMVPS 81 PTSSEEPCFP GCLTWLLENQ CRDGSWARPH HHSLLKKDVL 121 SSTLACILAL KKWGVCEEQI NKGLHFIELN CASATEKCQI 161 TPVGFDIIFP AMLDYARDFS LNLRLEPTTF NDLMDKRDLE 201 LKRCYQNYTP EREAYLAYIV EGMGRLQDWE LVMKYQRKNG 241 SLFNCPSTTA AAFIALRDSA CLNYLNLSLK KFGNAVPAVY 281 PLDIYSQLCT VDNLERLGIN QYFIAEIQSV LDETYRCWIQ 321 GNEDIFLDTS TCALAFRILR MNGYDVTSDS TTKILEECFS 361 SSFRGNMTDI NTTLDLYRAS ELMLYPDEKD LEKHNLRLKL 401 LLKQKLSTVL IQSFQLGRNI NEEVKQTLEH PFYASLDRIA 441 KRKNIEHYNF DNTRILKTSY CSPNFGNKDF FFLSIEDFNW 481 CQVIHRQELA ELERWLIENR LDELKFARSK SAYCYFSAAA 521 TFFAPELSDA RMSWAKSGVL TTVVDDFFDV GGSMEELKNL 561 IQLVELWDVD ASTKCSSHNV HIIFSALRRT IYEIGNKGFK 601 LQGRNITNHI IDIWLDLLNS MMKETEWARD NFVPTIDEYM 641 SNAYTSFALG PIVLPTLYLV GPKLSEEMIN HSEYHNLFKL 681 MSTCGRLLND IRGYERELKD GKLNALSLYI INNGGKVSKE 721 AGISEMKSWI EAQRRELLRL VLESNKSVLP KSCKELFWHM 761 CSVVHLFYCK DDGFTSQDLI QVVNAVIHEP IALKDFKVHE A nucleic acid encoding the Nepeta mussinii ent-kaurene synthase (NmTPS2) with SEQ ID NO:7 is shown below as SEQ ID NO:8.

1 ATGTCTCTIC CGCTCTCCTC TTGTGTCTTA TTTCCCCCCA 41 ATGACTCACG TTTTCCGCTC TCCCGCTTTT CTCGCGCTTC 81 AGCTTCTTTG GAAGTCGGGC TTCAAGGAGC TACTTCAGCA 121 AAAGTCTCCT CACAATCATC GTGTTTTGAG GAGACAAAGA 161 GAAGGATAAC AAAGTTGTTT CATAAGGACG AACTTTCGGT 201 TTCGACATAT GACACAGCAT GGGTTGCTAT GGTCCCTTCT 241 CCAACTTCTT CAGAGGAACC TTGCTTCCCA GGTTGTTTGA 281 CTTGGTTGCT TGAAAACCAG TGTCGAGATG GTTCATGGGC 321 TCGTCCCCAC CATCACTCTT TGTTAAAAAA AGATGTCCTT 361 TCTTCTACCT TGGCATGCAT TCTCGCACTT AAAAAATGGG 401 GGGTTGGTGA AGAACAAATC AACAAGGGTT TGCATTTTAT 441 AGAGCTAAAT TGTGCTTCAG CTACCGAGAA GTGTCAAATT 481 ACTCCCGTGG GGTTTGACAT TATATTTCCT GCCATGCTTG 521 ATTATGCAAG AGACTTCTCT TTGAACTTGC GTTTAGAGCC 561 AACTACGTTT AATGATTTGA TGGATAAAAG GGATTTAGAG 601 CTCAAAAGGT GTTACCAAAA TTACACACCG GAGAGGGAAG 641 CATACTTGGC ATATATAGTT GAAGGAATGG GAAGATTGCA 681 AGATTGGGAA TTGGTGATGA AATATCAAAG AAAGAATGGA 721 TCTCTTTTCA ATTGTCCATC TACAACTGCA GCAGCTTTTA 761 TTGCCCTTCG GGATTCTGCG TGCCTCAACT ATCTGAATTT 801 GTCTTTGAAA AAGTTCGGGA ATGCAGTTCC TGCAGTTTAT 841 CCTCTAGATA TATATTCTCA ACTTTGCACG GTTGATAATC 881 TTGAAAGGCT GGGGATCAAC CAATATTTTA TAGCAGAAAT 921 TCAGAGTGTG TTGGATGAAA CGTACAGATG TTGGATACAG 961 CGAAACGAAG ACATATTTTT GGACACCTCA ACTTGTCCTT 1001 TAGCATTCCG AATATTGAGA ATGAATGGCT ATGATGTGAC 1041 TTCAGATTCA CTTACAAAAA TCCTAGAAGA GTGCTTTTCA 1081 AGTTCCTTTC GTGGAAATAT GACAGACATT AACACAACTC 1121 TTGACTTATA TAGGGCATCA GAACTTATGT TATATCCAGA 1161 TGAAAAGGAT CTGGAGAAAC ATAATTTAAG GCTTAAACTC 1201 TTACTTAAGC AAAAACTATC CACTGTTTTA ATCCAATCAT 1241 TTCAACTTGG AAGAAATATC AATGAAGAGG TGAAACAGAC 1281 TCTCGAGCAT CCCTTTTATG CAAGTTTGGA TAGGATTGCA 1321 AAGCGGAAAA ATATAGAGCA TTACAACTTT GATAACACAA 1361 GAATTCTTAA AACTTCATAT TGTTCGCCAA ATTTTGGCAA 1401 CAAGGATTTC TTTTTTCTTT CCATAGAAGA CTTCAATTGG 1441 TGTCAAGTCA TACATCGACA AGAACTCGGA GAACTTGAAA 1481 GATGGTTAAT TGAAAATAGA TTGGATGAGC TGAAGTTTGC 1521 AAGGAGTAAG TCTGCATACT GTTATTTTTC TGCGGCAGCA 1561 ACTTTTTTIG CTCCAGAATT GTCGGATGCC CGCATGTCAT 1601 GGGCTAAAAG TGGTGTTCTA ACCACAGTGG TAGATGACTT 1641 TTTTGATGTT GGAGGTTCTA TGGAGGAATT GAAGAACTTA 1681 ATTCAATTGG TTGAACTATG GGATGTGGAT GCTAGCACAA 1721 AATGCTCTTC TCATAATGTC CATATAATAT TTTCAGCACT 1761 TAGGCGCACC ATCTATGAGA TAGGGAACAA AGGATTTAAG 1801 CTACAAGGAC GTAACATTAC CAATCATATA ATTGACATTT 1841 GGCTAGATTT ACTAAACTCT ATGATGAAAG AAACCGAATG 1881 GGCCAGAGAC AACTTTGTCC CAACAATTGA TGAATACATG 1921 AGCAATGCAT ATACATCGTT TGCTCTGGGG CCAATTGTCC 1961 TTCCAACTCT CTATCTTGTC GGGCCCAAGC TCTCAGAAGA 2001 GATGATTAAC CACTCCGAAT ACCATAACCT ATTCAAATTG 2041 ATGAGTACGT GCGGACGTCT TCTAAATGAC ATCCGTGGTT 2081 ATGAGAGAGA ACTGAAAGAT GGTAAATTGA ACGCGTTATC 2121 ATTGTACATA ATTAATAATG GTGGTAAAGT AAGTAAAGAA 2161 GCTGGCATCT CGGAGATGAA AAGTTGGATC GAGGCACAAC 2201 GAAGAGAGTT ACTGAGATTA GTTTTGGAGA GCAACAAAAG 2241 CGTCCTTCCG AAGTCGTGCA AGGAATTGTT TTGGCATATG 2281 TGCTCAGTGG TGCATCTATT CTACTGCAAA GATGATGGAT 2321 TCACCTCGCA GGATTTGATT CAAGTTGTAA ATGCAGTTAT 2361 TCATGAACCT ATTGCTCTCA AGGATTTTAA GGTGCATGAA 2401 TAA

10 An Origanum majorana trans-abienol synthase (OmTPS3) was identified and isolated. When this OmTPS3 enzyme was expressed in N. benthamiana with Hyptis suaveolens labda-7,13E-dienyl diphosphate synthase (HsTPS1) a new compound, labda-7,12E,14-triene [24], was produced. The HsTPS1 enzyme produced labda-7,13(16), 14-triene [22] when HsTPS1 was expressed in N. benthamiana.

OmTPS3 also produced trans-abienol [11] from labda-13-en-8-ol diphosphate ((+)-8-LPP) [10]).

The Origanum majorana trans-abienol synthase (OmTPS3) has the amino acid sequence shown below (SEQ ID NO:9).

MASLAFTPGA ATFSGNVVRR RKDNFPVHGF PTTIRSSVSV TVKCYVSTTN LMVNIKEKFK GKNVNSLTVE AADDDMPSNL CIIDTLQRLG IDRYFQPQVD SVLDHAYKLW QGKEKDTVYS DISIHAMAFR LLRVKGYQVS SEELDPYIDV ERMKKLKTVD VPTVIELYRA AQERMYEEEG SLERLHVWST NFLMHQLQAN SIPDEKLHKL VEYYLKNYHG ILDRVGVRRN LDLFDISHYP TLRARVPNLC TEDFLSFAKE DFNTCQAQHQ KEHEQLQRWF EDCRFDTLKF GRETAVGAAH FLSSAILGES ELCNVRLALA KHMVLVVFID DFFDHYGSRE DSFKILHLLK EWKEKPAGEY GSEEVEILFT AVYNTVNELA EMAHVEQGRN IKGFLIELWV EIVSIFKIEL DTWSNDTTLT LDEYLSSSWV SVGCRICILV SMQLLGVQLT DEMLLSDECI NLCKHVSMVD RLLNDVGTFE KERKENTGNS VSLLLAAAVK EGRPITEEEA IIKIKKMAEN ERRKLMQIVY KRESVFPRKC KDMFLKVCRI GCYLYASGDR FTSPQKMKED VKSLIYESL A nucleic acid encoding the Origanum majorana trans-abienol synthase (OmTPS3) with SEQ ID NO:9 is shown below as SEQ ID NO: 10.

ATGGCGTCGC TCGCGTTCAC ACCCGGAGCC GCCACTTTCT CCCCCAACCT AGTTCGGAGG AGGAAAGATA ACTTTCCGGT CCACGGATTT CCGACGACGA TCAGGTCATC GGTCTCCGTC ACCGTCAAAT GCTACGTCAG TACAACGAAT TTGATGGTGA AAATCAAAGA GAAGTTCAAG GGTAAAAACG TCAATTCGCT GACAGTTGAA GCTGCTGATG ACGATATGCC CTCTAATCTG TGCATAATTG ACACCCTCCA ACGATTGGGA ATCGACCGTT ACTTCCAACC CCAACTCGAC TCTGTTCTCG ACCACGCCTA CAAACTATGG CAAGGGAAAG AGAAAGATAC GGTGTATTCG GACATTAGTA TTCATGCGAT GGCATTTAGA CTTTTACGAG TCAAAGGCTA TCAAGTCTCT TCGGAGGAAC TGGATCCATA CATCGATGTG GAGCGAATGA AGAAACTGAA AACAGTTGAT GTTCCGACGG TTATCGAACT GTACAGAGCG GCACAGGAGA GAATGTATGA AGAAGAAGGT AGCCTTGAGA GACTCCATGT TTGGAGCACC AACTTCCTCA TGCACCAGCT GCAGGCTAAC TCAATTCCTG ATGAAAAGCT ACACAAACTG GTGGAATACT ACTTGAAGAA CTACCATGGC ATACTGGATA GAGTTGGAGT TCGACGAAAC CTCGACCTAT TCGACATAAG CCATTATCCA ACACTCAGAG CTAGGGTTCC GAACCTATGT ACCGAAGATT TTCTATCGTT CGCGAAGGAA GATTTCAATA CTTGCCAAGC CCAACACCAG AAAGAACATG AGCAACTACA AAGGTGGTTC GAAGATTGTA GGTTCGATAC GTTGAAGTTC GCAAGGGAGA CAGCCGTAGG CGCTGCTCAT TTTCTATCTT CAGCAATACT TGGTGAATCT GAACTATGTA ATGTTCGTCT TGCCCTTGCT AAGCATATGG TGCTTGTGGT ATTCATCGAT GACTTCTTCG ACCATTATGG CTCTAGAGAA GACTCCTTCA AGATCCTCCA CCTCTTAAAA GAATGGAAAG AGAAGCCGGC CGGAGAATAC GGTTCCGAGG AAGTCGAAAT CCTCTTCACA GCCGTATACA ATACAGTAAA CGAGTTGGCG GAGATGGCTC ATGTCGAACA AGGACGTAAT ATCAAAGGAT TTCTAATTGA ATTGTGGGTT GAAATAGTGT CAATTTTCAA GATAGAACTG GATACATGGA GCAATGACAC AACACTAACC TTGGATGAGT ACTTGTCCTC CTCATGGGTG TCGGTCGGTT GCAGAATCTG CATCCTCGTC TCAATGCACC TCCTCGGTGT ACAACTAACC GACGAAATGC TTCTGAGCGA CGAGTGCATA AACCTGTGTA AGCATGTCTC GATGGTCGAT CGCCTCCTCA ACGACGTCGG AACATTCGAG AAGGAACGGA AGGAGAATAC AGGAAACAGT GTGAGCCTTC TGCTAGCAGC AGCTGTGAAA GAAGGAAGGC CTATTACCGA AGAGGAAGCT ATTATTAAAA TTAAAAAAAT GGCGGAAAAC GAGAGGAGGA AACTAATGCA GATTGTGTAT AAAAGAGAGA GTGTTTTCCC CAGAAAATGC AAGGATATGT TCTTGAAGGT GTGTAGAATT GGGTGCTATC TATACGCGAG CGGCGACGAA TTTACGTCTC CTCAGAAAAT GAAGGAAGAT GTGAAATCCT TAATTTATGA ATCCTTGTAG

The Origanum majorana manool synthase (OmTPS4) can also convert ent-copalyl diphosphate (ent-CPP) [16] to ent-manool [20].

In addition, Origanum majorana manool synthase (OmTPS4) can also convert (+)-copalyl diphosphate ((+)-CPP) [31]) to manool [33].

The Origanum majorana manool synthase (OmTPS4) can have the amino acid sequence shown below (SEQ ID NO:11).

MSLAFSHVST FFSGQRVVGS RREIIPVNGV PTTANKPSFA VKCNLTTKDL MVKMKEKLKG QDGNLTVGVA DMPSSLCVID TLERLGVDRY FRSEIHVILH DTYRLWQQKD KDICSNVTTH AMAFRLLRVN GYEVSSEELA PYANLEHFSQ QKVDTAMAIE LYRAAQERIH EDESGLDKIL AWTTTFLEQQ LLTNSILDNK LHKLVEYYLN NYHGQTNRVG ARRHLDLYEM SHYQNLKPSH SLCNEDLLAF AKQGFRDFQI QQQKEFEQLQ RWYEDCRLDK LSYGRDVVKI SSFMASILMD DPELADVRLS IAKQMVLVTR IDDFFDHGGS REDSYKIIEL VKEWKEKAEY DSEEVKILFT AVYTTVNELA EACVQQGRNS TTVKEFLVQL WIEILSAFKV ELDTWSDGTE VSLDEYLSWS WISNGCRVSI VTTMHLLPTK LCSDEMLRSE ECKDLCRHVS MVCRLLNDIH SFEKEHEENT GNSVSILVAG EDTEEEAIGK IKEIVEYERR KLMQIVYKRG TILPRECKDI FLKACRATFY VYSSTDEFTS PRQVMEDMKT LSS A nucleic acid encoding Origanum majorana manool synthase (OmTPS4) with SEQ ID NO:11 is shown below as SEQ ID NO: 12.

ATGTCACTCG CCTTCAGCCA TGTTAGTACC TTTTTCTCCC GCCAAAGAGT CGTCGGAAGC AGGAGAGAGA TTATTCCAGT TAACCGAGTT CCGACGACGG CCAATAAGCC GTCGTTCGCC GTTAAGTGCA ACCTTACTAC AAAGGATTTG ATGGTGAAAA TGAAGGAGAA GTTGAAGGGG CAAGACCGTA ATTTGACTGT CGGAGTAGCC GATATGCCCT CTAGCCTGTG CGTGATCGAC ACTCTTGAAA GGTTGGGAGT TGACCGATAC TTCCGATCTG AAATCCACGT TATTCTACAC GACACTTACC GGTTATGGCA ACAAAAGGAC AAAGATATAT GTTCCAACGT TACTACTCAT GCAATGGCGT TTAGACTTCT GAGAGTGAAT GGATACGAGG TTTCATCAGA GGAACTGGCT CCATATGCTA ACCTAGAGCA CTTTAGCCAG CAAAAAGTTG ATACTGCAAT GGCTATAGAG CTCTACAGAG CAGCACAGGA GAGAATACAC GAAGACGAGA GCGGTCTCGA CAAAATACTT GCTTGGACCA CCACTTTTCT CGAGCAACAG CTGCTCACTA ACTCCATTCT TGACAATAAA TTGCATAAAC TGGTGGAGTA CTACTTGAAC AACTACCACG CCCAAACGAA TAGGGTCGGA GCTAGACGAC ACCTCGACCT ATATGAGATG AGCCATTACC AAAATCTAAA ACCTTCACAT AGTCTATGCA ATGAAGACCT TCTAGCATTT GCAAAGCAAG GTTTTCGAGA TTTTCAAATC CAGCAGCAGA AAGAATTCGA GCAACTGCAA AGGTGGTATG AAGATTGCAG GTTGGACAAG TTGAGTTATG GGAGAGATGT AGTAAAAATT TCTAGTTTCA TGGCTTCAAT ATTGATGGAT GATCCAGAAT TAGCCGATGT TCGTCTCTCC ATCGCCAAAC AGATGGTGCT CGTGACACGT ATCGATGATT tCTTCGACCA CGGTGGCTCT ACAgAaGACT CCTACAAGAT CATTGAACTA GTAAAAGAAT GGAAGGAGAA GGCaGAATAC GATTCCGAGG AAGTAAAAAT CCTTTTTACA GCAGTATACA CCACAGTAAA TGAGCTAGCA GAGGCTTGTG TTCAACAAGG AAGGAATAGT ACTACTGTCA AAGAATTCCT AGTTCAGTTG TGGATTGAAA TACTATCAGC TTTCAAGGTC GAGCTAGATA CGTGGAGCGA TGGCACGGAA GTAAGCCTGG ACGAGTACTT GTCGTGGTCG TGGATTTCGA ATGGCTGCAG AGTGTCTATA GTAACGACGA TGCATTTGCT CCCTACGAAA TTATGCAGTG ATGAAATGCT TAGGAGTGAA GAGTGCAAGG ATTTGTGTAG GCATGTTTCT ATGGTTGGCC GCTTGCTCAA CGACATCCAC TCTTTTGAGA AGGAGCATGA GGAGAATACG GGAAACAGTG TGAGCATTCT AGTAGCAGGT GAGGATACCG AAGAGGAAGC TATTGGAAAG ATCAAAGAGA TAGTTGAGTA TGAGAGGAGA AAATTGATGC AAATTGTGTA CAAGAGAGGA ACCATTCTCC CAAGAGAATG CAAAGACATA TTCTTGAAGG CGTGTAGGGC TACATTTTAC GTGTACTCGA GCACGGATGA GTTTACGTCT CCTCGACAAG TGATGGAAGA TATGAAAACC CTAAGCTCCT AG

Origanum majorana palustradiene synthase (OmTPS5) can also convert (+)-copalyl diphosphate ((+)-CPP) [31]) to palustradiene [29].

The Origanum majorana palustradiene synthase (OmTPS5) can have the amino acid sequence shown below (SEQ ID NO: 13).

MVSACLKLKN NPFLDHRFRK SSNGFSVNFP ATMLTTVKCS RDNSEDLIAK IKERMNEKFV TVPAREYSVI EHRNPKPAWC GGLQSKTVIE EEVCSRLFLV EHLQDLGVDR FFQSEIQHIL HHTFRLWQQK DEQVFKDVTC RAMAFRLLRL EGYHVSSGEL GEYVDEEKFF RTVRLEWRST DTILELYKAS QVRLPEDDND NSNILKNLHE WTFIFLKEQL RRKTILDKGL ERKVEFYLKN YHGILDAVKH RRSLDHTRFW KTTAYNPAVY DEDLERLSAQ DFMARQAQSQ KELEMLLKWY DECRLDKMEY GRNVIHVSHF LNANNFPDPR LSETRLSFAK TMTLVTRLDD FFDHHGSRED SVLIIELIRQ WNEPSTITTI FPSEEVEILY SALHSTVTDI AEKAYPIQGR CIKSLIIHLW VEILSSFMSE MDSCTAETQP DFHEYLGFAW ISIGCRICIL IAIHFLGEKV SQQMVMGAEC TELCRHVSTI ARLLNDLQTF KKEREERKVN SVIIQLKGDK ISEEVAVSNI ERMVEYHRKE LLKMVVRREG SLVPKRCKDV FWKSCNIAYY LYAFTDEFTS PQQMKEDMKL LFRDPINCVP SIPS A nucleic acid encoding the Origanum majorana palustradiene synthase (OmTPS5) with SEQ ID NO:13 is shown below as SEQ ID NO:14.

ATGGTATCTG CATGTCTAAA ACTCAAAAAT AATCCTTTCT TGGACCATCG ATTCAGGAAA AGCAGCAATG GATTTTCAGT TAATTTTCCG GCGACCATGC TCACCACTGT CAAGTGCAGC CGCGATAATT CAGAAGACTT GATAGCAAAG ATAAAAGAAA GGATGAATGA AAAATTTGTT ACGGTGCCGG CGAGGGAATA TTCCGTCATT GAGCATCGGA ATCCGAAGCC GGCGTGGTGC GGTCGTITGC AATCCAAAAC AGTAATAGAA GAAGAAGTGT GCAGCCGTCT GTTTCTGGTC GAACACCTTC AAGATTTAGG AGTAGACCGC TTCTTTCAAT CAGAAATCCA ACATATTCTA CATCACACAT TCAGATTATG GCAGCAAAAA GATGAACAAG TTTTTAAAGA CGTGACATGT CGCGCCATGG CATTCAGACT CCTGCGTCTC GAAGGTTATC ATGTCTCGTC AGGAGAATTG GGGGAGTATG TTGATGAGGA AAAATTCTTT AGAACGGTAA GGTTAGAATG GAGAAGTACG GATACAATTC TTGAGCTGTA CAAAGCATCA CAGGTAAGAC TACCTGAAGA CGACAACGAC AATTCCAATA TCCTCAAAAA CTTGCACGAA TGGACCTTCA TATTTTTGAA GGAGCAGTTG CGGCGTAAAA CTATTCTTGA TAAAGGTTTA GAGAGAAAGG TAGAATTTTA CTTGAAGAAT TACCACGGCA TATTAGACGC GGTTAAGCAT AGACGAAGCC TCGATCACAC ACGATTCTGG AAAACTACTG CGTATAACCC TGCAGTGTAT GATGAGGATC TTTTCCGATT GTCGGCCCAA GATTTCATGG CTCGCCAAGC TCAGAGCCAG AAGGAACTTG AGATGTTGCT CAAGTGGTAC GATGAATGTA GACTGGACAA GATGGAGTAT GGGCGAAACG TGATACACGT TTCCCATTTC TTAAACGCAA ACAACTTCCC CGATCCTCGC CTGTCCGAAA CTCGTCTATC CTTTGCGAAA ACCATGACTC TCGTCACGCG TTTGGATGAT TTCTTCGATC ACCATGGCTC TAGAGAAGAT TCGGTCCTCA TCATCGAATT AATAAGGCAG TGGAATGAGC CTTCAACTAT TACAACAATA TTCCCCTCCG AAGAAGTGGA GATTCTCTAC TCTGCACTCC ACTCCACCGT AACAGATATA GCAGAGAAGG CTTATCCCAT CCAGGGTCGC TGCATCAAAT CGCTCATAAT TCATCTGTGG GTCGAGATAC TGTCGAGCTT CATGAGCGAA ATGGACTCGT GCACCGCGGA AACTCAGCCG GACTTTCACG AGTACTTAGG GTTTGCATGG ATCTCGATCG GCTGCAGAAT CTGCATTCTC ATAGCTATAC ATTTCTTGGG GGAGAAGGTA TCTCAACAAA TGGTTATGGG TGCTGAGTGC ACCGAGTTAT GTAGGCACGT TTCTACGATC GCACGCCTTC TCAACGATCT CCAAACCTTT AAGAAGGAGA GAGAAGAGAG GAAGGTAAAC AGCGTGATAA TCCAGCTCAA AGGGGATAAG ATATCGGAGG AGGTGGCCGT GTCGAATATA GAGAGAATGG TTGAATATCA CAGGAAAGAG CTGCTGAAGA TGGTGGTTCG GAGAGAAGGA AGCTTGGTTC CTAAGAGGTG TAAGGACGTG TTCTGGAAAT CCTGCAACAT TGCTTACTAT CTGTACGCTT TTACAGATGA ATTCACTTCG CCTCAACAAA TGAAGGAAGA TATGAAACTA CTCTTTCGTC ATCCAATCAA CTGCGTTCCT TCAATTCCTT CATGA

The Perovskia atriplicifolia miltiradiene synthase (PaTPS3) can have the amino acid sequence shown below (SEQ ID NO: 15).

MLLAFNISDV PLSQHRVILS RREHFPRHAF QEFPMIAATK SSVNAICSLA TPIDLMGKIK EKFKAKDGDP LAAAAIQLAA DIPSSLCITD TLQRLGVDRY FQSEIDSILE ETHKLWKVKD RDIYSEVTTH AMAFRLLRVK GYEVSSEELA PYAEQERFDL QTIDLATVIE LYRAAQERTC EENDNSLEKL LAWTTTFLKH QLLTNSIPDT KLHKQVEYYL KNYHGILDRM GVRRSLDLYD ISHYRPLRAR FPNLCNEDFL SFARQDFSMC QAQHQKELEQ LQRWYSDCRL DALLKFGRNV VRVSSFLTSA IIGEPELSEV RLVFAKHIIL VTLIDDLFDH GGTREESYKI LELVTEWKEK TAAEYGSEEV EILETAVYNT VNELVERAHV EQGRSVKEFL IKLWVQILSI FKIELDTWSD ETALTLDEYL SSSWVSIGCR ICILMSMQFI GIKLTDEMLL SEECTDLCRH VSMVDRLLND VQTFEKERKE NTGNSVSLLL AANKDVTEEE AIRRAKEMAE CNRRQLMQIV YKTGTIFPRK CKDMFLKVCR IGCYLYASGD EFTSPQQMME DMKSLVYEPL YLPN A nucleic acid encoding the Perovskia atriplicifolia miltiradiene synthase (PaTPS3) with SEQ ID NO: 13 is shown below as SEQ ID NO: 16.

ATGTTACTTG CGTTCAACAT AAGCGATGTC CCTCTCTCGC AGGATAGAGT AATTCTGAGC AGGAGGGAAC ATTTTCCACC TCATGCATTC CAGGAATTTC CGATGATCGC CGCTACTAAG TCATCTGTTA ATGCCATTTG CAGCCTCGCT ACTCCAACTG ATTTGATGGG AAAAATAAAA GAGAAGTTCA AGGCCAAGGA CGGCGATCCT CTTGCCGCCG CGGCTATTCA ACTCGCGGCG GATATACCCT CGAGTCTGTG TATAATCGAC ACCCTCCAGA GGTTGGGAGT CGACCGATAC TTCCAATCCG AAATCGACTC TATTCTAGAG GAAACACACA AGTTATGGAA AGTGAAAGAT AGAGATATAT ACTCTGAGGT TACTACTCAT GCAATGGCGT TTAGACTTCT GCGAGTGAAG GGATATGAAG TTTCATCAGA CGAACTACCT CCGTATGCTC ACCAAGAGCG CTTTGACCTG CAAACGATTG ATCTGGCGAC GGTTATCGAG CTTTACAGAG CAGCACAGGA GAGAACATGC GAAGAAAACG ACAACAGTCT TGAGAAACTA CTTGCTTGGA CCACCACCTT TCTCAAGCAC CAATTGCTCA CCAACTCCAT ACCTGACACC AAATTGCACA AACAGGTGGA ATACTACTTG AAGAACTACC ACGGGATATT AGATAGAATG GGAGTTAGAC GAAGCCTCGA CCTATACGAC ATAAGCCATT ATCGACCTCT GAGAGCAAGA TTCCCTAATC TGTGTAATGA AGATTTCCTA TCATTTGCGA GGCAAGATTT CAGTATGTGC CAACCCCAAC ACCAGAAGGA ACTTGAGCAA CTGCAAAGGT GGTATTCTGA TTGTAGGTTG GACGCGTTGT TGAAGTTTGG AAGAAATGTA GTGCGCGTTT CTAGCTTTCT GACTTCAGCA ATTATTGGTG AACCCGAATT GTCTGAAGTT CGACTAGTCT TTGCCAAACA TATTATTCTC GTTACACTTA TTCATGATTT ATTCGATCAT GGTGGAACTA GAGLAGAGTC ATACAAGATC CTTGAATTAG TAACAGAATG GAAAGAGAAG ACCGCAGCAG AATATGGTTC CGAGGAAGTT GAAATCCTTT TTACAGCGGT CTACAACACA GTAAATGAGT TGGTAGAGAG GGCTCATGTC GAACAAGGGC GCAGTGTCAA AGAATTTCTT ATTAAACTGT GGGTTCAAAT ACTATCAATT TTCAAGATAG AATTAGATAC ATGGAGCGAT GAGACTGCGC TAACCTTGGA TGAATACTTG TCTTCGTCGT GGGTGTCAAT TGGTTGCAGA ATCTGCATTC TCATGTCGAT GCAATTCATC GGTATAAAAT TAACTGATGA AATGCTTCTG AGTGAAGAGT GCACTGATTT GTGTAGGCAT GTTTCGATGG TTGACCGGCT GCTCAACGAT GTGCAAACCT TCGAGAAGGA ACGCAAAGAA AATACAGGAA ACAGTGTAAG CCTTCTGCTA GCAGCTAACA AAGATGTTAC TGAAGAGGAA GCAATTAGAA GAGCAAAAGA AATGGCGGAA TGCAACAGGA GACAACTGAT GCAGATTGTG TATAAAACAG GAACCATTTT CCCAAGAAAA TGCAAAGATA TGTTTCTCAA GGTATGCAGG ATTGGCTGTT ATTTGTATGC AAGCGGCGAC GAATTCACAT CTCCACAACA AATGATGGAA GATATGAAAT CCTTCGTTTA TGAACCCCTC TACCTACCTA ATTAA

A Perovskia atriplicifolia miltiradiene synthase (PaTPS1) can have the amino acid sequence shown below (SEQ ID NO:17).

MSLTFNAGVV RFSSHRVRST KDCFTVYGFP MIANKAAFAV KCSLTPTDLM GRVEEKFKGK NGNSLAASTT VESADIPSNL CIIDTLQRLG VDRYFQTEIN AILEDTYRLW ERKDKDIYSD ATTHAMAFRL LRVKGYEVSS EELAPYADQE CVNVQTADVA TVIELYRAAQ VRISEEESSL KKLHAWTTTF LKYQLQSNSI PEKKLHKLVE YYLKNYHGIL DRMGVRMDLD LFDISHYRTL QASDRFSSLR NEDFLEFARQ DFNICQAKHQ KELQQLQRWY ADCRLDTLKF GRDVVRVANF LTSAIFGEPE LSDARLIFAK HIVLVTCIDE FFDHGGSKEE SYKILELVEE WKEKPTGEYG CEEVEILFTA VYSTVNELAE MAHVEQGRSV KEFLVKLWVQ ILSIFKIELD TWSDDTELTL DSYLNNSWVS IGCRICILMS MQFAGVKLSD EMLLSEECVD LCRHVSMVDR LLNDVQTFEK ERKENTGNSV SLLQAAAERE GRAITEEEAI TQIKELAEYH RRKLMQIVYK TDTIFPRKCK DMFLKVCRIG CYLYASGDEF TTPQQMMEDM KSLVYQPLTV DDMSAKELTS VRN A nucleic acid encoding the Perovskia atriplicifolia miitiradiene synthase (PaTPS1) with SEQ ID NO: 13 is shown below as SEQ ID NO: 18.

ATGTCACTCA CTTTCAACGC TGGAGTCGTC CGTTTCTCCA GCCACCGCGT TCGGAGCACG AAAGATTGCT TTACAGTTTA CCGATTTCCG ATGATTGCAA ATAAGGCAGC TTTCGCAGTT AAATGCAGCC TTACTCCAAC CGATTTGATG GGGAGAGTAG AGGAGAAGTT CAAGGGCAAA AATGGTAATT CACTAGCAGC CTCGACGACG GTTGAATCCG CGGATATACC CTCGAACCTG TGTATAATCG ACACCCTCCA AAGATTGGGA GTCGACCGAT ACTTTCAAAC TGAAATCAAT GCCATTCTAG AGGACACTTA CAGATTATGG GAACGAAAAG ACAAAGACAT ATATTCCGAT GCCACAACTC ACGCGATGGC GTTTAGGTTA CTACGAGTGA AAGGATACGA AGTTTCATCA GAGGAACTGG CTCCTTACGC TGATCAAGAG TGCGTGAACG TGCAAACGGC TGATGTGGCA ACAGTTATCG AGCTTTACAG AGCAGCGCAG GTGAGAATAA GCGAAGAAGA GAGCAGTCTT AAGAAGCTTC ATGCTTGGAC CACCACCTTT CTCAAATATC AGTTGCAGAG TAACTCCATA CCTGAAAAGA AACTGCACAA ACTGGTGGAA TATTACTTGA AGAACTACCA TGGCATATTG GATAGAATGG GAGTTCGAAT GGACCTCGAC TTATTCGACA TCAGCCATTA TCGAACTCTA CAAGCTTCCG ATAGGTTCTC TAGTCTGCGT AACGAAGATT TTCTAGAGTT TGCAAGGCAA GATTTCAATA TCTGCCAAGC CAAGCACCAG AAAGAACTCC AACAACTGCA AAGGTGGTAT GCAGATTGCA GGCTCGACAC CTTGAAGTTC GGGAGAGACG TCGTACGCGT TGCTAATTTT CTGACTTCAG CAATCTTTGG CGAACCCGAG CTATCCGATG CTCGTCTGAT CTTTGCCAAG CATATCGTGC TCGTAACATG TATCGATGAA TTCTTCGATC ATGGTGGGTC TAAAGAAGAG TCCTACAAGA TCCTTGAATT AGTAGAAGAA TGGAAAGAGA AGCCAACTGG AGAATATGGG TGTGAGGAGG TTGAGATCCT TTTCACAGCA GTGTACAGTA CAGTGAATGA GTTGGCAGAG ATGGCTCATG TCGAACAAGG ACGTAGTGTG AAAGAGTTTC TAGTTAAACT GTGGGTGCAG ATACTGTCGA TTTTCAAGAT AGAACTGGAT ACATGGAGTG ATGACACGGA ACTGACGTTG GACAGCTACT TGAACAACTC GTGGGTGTCG ATCGCATGCA GAATCTGCAT TCTCATGTCG ATGCAGTTCG CCGGTGTAAA ACTGTCCGAC GAAATGCTTC TGAGTGAAGA GTGTGTTGAC TTGTGCAGGC ACGTCTCCAT GGTCGATCGC CTCCTGAACG ATGTGCAAAC TTTCGAGAAG GAACGCAAGG AAAATACAGG AAACAGTGTG AGCCTTCTGC AAGCAGCAGC TGAGAGAGAA GGAAGACCCA TTACAGAAGA GGAAGCTATT ACACAGATCA AAGAATTGGC TGAATACCAC AGGAGAAAAC TGATGCAGAT TGTGTACAAA ACAGACACCA TTTTCCCAAG AAAATGCAAA GATATGTTCT TGAAGGTGTG CAGGATTGGG TGCTATCTGT ACGCAAGTGG AGACGAATTC ACAACTCCAC AACAAATGAT GGAAGACATG AAATCATTGG TTTATCAACC CCTAACAGTT GATGACATGA GTGCCAAAGA ATTGACTTCT GTGAGAAACT AG

The Salvia officinalis miltiradiene synthase (SoTPS1) can have the amino acid sequence shown below (SEQ ID NO: 19).

MSLAFNAAVA TFSGHRIRSR REILPGQGFP MITNKSSFAV KCNLTTIDLM GKITEKFKGR DSNFSAATAV QPAADIPSNL CIIDTLQRLG VDRYFQSEID TILEDTYRLW QRKEREIFSD ITIHAMAFRL LRVKGYVVSS EELAPYADQE RINLQRIDVA TVIELYRAAQ ERISEDESSL EKLHAWTATY LKQQLLTNSI PDKKLNKLVE CYLKNYHGIL DRMGVRQNLD LYDISHYQTL KAADRFSNLR NEDFLAFARQ DFNICQEQHQ KELQQLQRWY ADCRLDTLKY GRDVVRVANF LTSAIIGDPE LSEVRLVFAK HIVLVTRIDD FFDHGGSREE SYKILELLKE WKEKPAAEYG SKEVEILFIA VYNTVNELAE MAHIEQGRSV KEFLIKLWVQ IISIFKIELD TWSDETALTL DEYLSSSWVS IGCRICILMS MQFIGIKLSD EMLLSEECID LCREVSMVDR LLNDVQTFEK ERKENTGNSV SLLLAANKDD SAFTEEEAIT KAKEMAECNR RQLMKIVYKT GTIFPRKCKD MFLKVCRIGC YLYASGDEFT SPQQMMEDMK SLVYEPLTVD PLEAKNVSGK A nucleic acid encoding the Salvia officinalis miltiradiene synthase (SoTPS1) with SEQ ID NO: 19 is shown below as SEQ ID NO:20.

ATGTCCCTCG CCTTCAACGC AGCAGTTGCC ACTTTCTCCG GCCACAGAAT TCGGAGCAGG AGAGAAATTC TTCCGGGGCA AGGATTTCCG ATGATCACCA ACAAGTCGTC TTTCGCCGTG AAATGTAACC TTACTACAAC AGATTTGATG GGCAAGATAA CAGAGAAATT CAAGGGAAGA GACAGTAATT TTTCAGCAGC AACCGCTGTT CAACCTGCGG CGGATATACC CTCTAACCTG TGCATAATCG ACACCCTCCA AAGGTTGGGA GTCGACCGAT ACTTCCAATC TGAAATCGAC ACTATTCTAG AGGACACATA CAGGTTATGG CAAAGGAAAG AGAGAGAGAT ATTTTCGCAT ATAACTATTC ATGCAATGGC ATTTAGACTT TTGCGAGTTA AAGGATATGT AGTTTCATCA GAGGAACTGG CTCCGTATGC TGACCAAGAG CGCATTAACC TGCAAAGGAT TGATGTAGCG ACAGTTATCG AGCTTTACAG AGCAGCACAG GAGAGAATAA GTGAAGACGA GAGCAGTCTT GAGAAACTAC ATGCTTGGAC CGCCACCTAT CTCAAGCAGC AGCTGCTCAC TAACTCCATT CCTGAGAAGA AATTGAACAA ACTGGTGGAA TGCTACTTGA AGAACTATCA CGGGATATTA GATAGAATGG GAGTTAGACA AAACCTCGAC CTCTACGACA TAAGCCACTA TCAAACTCTA AAAGCTGCAG ATAGGTTCTC TAATCTACGT AATGAAGATT TTCTAGCATT TGCGAGGCAA GATTTTAATA TTTGCCAAGA ACAACACCAA AAAGAACTTC AGCAACTGCA AAGGTGGTAT GCAGATTGTA GGTTGGACAC ATTGAAGTAT GGAAGAGATG TCGTGCGGGT TGCTAATTTT CTAACATCAG CAATTATTGG TGATCCTGAA TTGTCTGAAG TCCGTCTAGT CTTCGCCAAA CATATTGTGC TTGTAACACG TATTGATGAT TTTTTCGATC ATGGTGGATC TAGAGAAGAG TCCTACAAGA TCCTTGAATT ACTAAAAGAA TGGAAAGAGA AGCCAGCTGC AGAATATGGT TCCAAAGAAG TTGAAATTCT TTTCACAGCA GTATACAATA CAGTAAACGA GTTGGCAGAG ATGGCTCACA TCGAACAAGG ACGTAGTGTT AAAGAATTTC TAATAAAGCT GTGGGTTCAA ATCATATCGA TTTTCAAGAT AGAATTAGAT ACATGGAGCG ATGAGACAGC GCTGACCTTG GATGAGTACT TGTCTTCGTC GTGGGTGTCA ATTGGGTGCA GAATCTGCAT TCTCATGTCG ATGCAATTCA TTGGTATAAA ATTATCTGAT GAAATGCTTC TGAGTGAAGA GTGTATTGAT TTGTGTCGGC ATGTCTCCAT GGTTGACCGG CTGCTCAACG ACGTGCAGAC TTTCGAGAAG GAACGCAAGG AAAATACAGG AAATAGCGTG AGCCTTCTGC TAGCAGCTAA CAAAGACGAC AGCGCCTTTA CTGAAGAGGA AGCTATTACA AAAGCAAAAG AAATGGCGGA ATGTAACAGG AGACAACTGA TGAAGATTGT GTATAAAACA GGAACCATTT TCCCAAGAAA ATGCAAAGAT ATGTTTCTGA AGGTATGCAG GATTGGCTGT TACTTGTATG CAAGCGGCGA TGAATTCACA TCTCCACAAC AAATGATGGA AGATATGAAA TCCTTGGTCT ATGAACCCCT AACAGTTGAT CCTCTCGAGG CCAAAAATGT GAGTGGCAAA TGA

Ajuga replans (+)-copalyl diphosphate synthase (ArTPS1) is a (+)-copalyl diphosphate ((+)-CPP) [31] synthase, and compound 31 is shown below.

The Ajuga reptans (+)-copalyl diphosphate synthase (ArTPS1) can have the amino acid sequence shown below (SEQ ID NO:21).

MASLSTFHLY SSSLLHRKTL QSSPKLNLSS ECFSTRTWMN SSKNLSLNYQ VNQKIGKLTG TRVATVDAPQ QLEHDDSTAK GHDIVDIETQ DPIEYIRMLL NTTGDGRISV SPYDTAWIAL IKDVEGRDFP QFPSSLEWIA NHQLADGSWG DEGFFCVYDR LVNTIACVVA LRSWNVHHDK SQRGIQYIKE NVHQLKDGNA EHMMCGFEVV FPALLQKAKN MGIDDLPYEA PVIQDIYHTR EQKLKRIPLE MMHKVPTSLL FSLEGLENLD WDKLLKLQSA DGSFLTSPSS TAFAFMQTKD EKCFQFIKNT VETFNGGAPH TYPVDVFGRL WAVDRLQRLG ISRFFEAEIA DCLSHIHRYW NDKGLFSGRE SDFVDIDDTS MGFRLLRMQG YDVSPNVLRN FKNGDKFSCY GGQTIESSTP IYNLYRASQF RFPGEEILEE ADKFAHEFLS EQLGNNQLLD KWVISDRLQE EISIGLGMPF YATLPRVEAS YYIQHYAGAD DVWIGKTLYR MPEISNDTYL ELARNDFKRC QAQHQFEWIY MQEWYESCNI EEFGISRKEL LRVYFLACSS IFEVERTKER MAWAKSQIIS RMITSFENKQ TTSSEEKETL LTEFRNINGL HKSNNTRDGD MNIVLATLHQ FFAGFDRYTS HQLKNAWGVW LSKLQRGAVD GGADAELITT TINVCAGHIA LKEDILSHDE YKTLTDLTSK ICQQLSHIQN EKVVEIDGGI TAKSRLKNEE LQRDMQSLVK LVLEKSVGLN RNIKQTFLTV AKTYYYRAYN AEETMDAHIF KVLFEPVA A nucleic acid encoding the Ajuga reptans (+)-copalyl diphosphate synthase (ArTPS1) with SEQ ID NO:21 is shown below as SEQ ID NO:22.

ATCGCCTCTT TGTCCACTTT CCACCTCTAC TCTTCCTCAC TCCTTCACCG CAAAACACTG CAATCTTCAC CAAAGCTTAA CCTGTCTTCA GAATGCTTCT CCACCAGAAC TTGGATGAAC AGCAGCAAAA ACTTGTCGTT AAATTACCAA GTTAATCAGA AAATAGGAAA GCTGACAGGG ACTCGAGTTG CCACTGTGGA TGCGCCACAA CAACTTGAAC ACGATGATTC AACTGCTAAA GGCCATGATA TAGTCGATAT TGAAACTCAG GATCCAATTG AATATATTAG AATGCTGTTG AACACAACAG GCGATGGCAG AATCAGCGTT TCGCCTTACG ACACAGCATG GATTGCTCTT ATTAAGGACG TGGAAGGACG TGATTTTCCT CAATTTCCAT CCAGCCTTGA GTGGATCGCG AACCATCAAC TCGCTGATGG TTCATGGGGA GACGAAGGAT TTTTCTGTGT GTATGATCGG CTCGTAAATA CTATAGCATG TGTCGTAGCA TTGAGATCAT CGAATGTCCA TCACGACAAG AGCCAAAGAG GAATACAATA TATCAAGGAA AATGTGCATC AACTTAAGGA TGGAAATGCT GAGCACATGA TGTGTGGTTT CGAAGTAGTG TTTCCTGCAC TTCTTCAAAA AGCCAAAAAT ATGGGCATTG ATGATCTTCC ATATGAGGCT CCTGTCATCC AGGATATTTA CCATACAAGG GAGCAGAAAT TGAAAAGGAT ACCATTGGAG ATGATGCACA AAGTGCCTAC TTCTCTGCTG TTTAGTTTGG AAGGACTGGA GAATTTAGAT TGGGATAAAC TCCTTAAGTT GCAGTCAGCT GATGGCTCTT TCCTCACTTC TCCCTCCTCT ACTGCTTTCG CATTCATGCA AACAAAAGAC GAAAAATGCT TCCAGTTCAT CAAGAACACT GTTGAAACCT TTAATGGAGG AGCACCACAT ACTTATCCGG TCGATGTTTT TGGAAGACTT TGGGCGGTTG ATAGGCTGCA GCGCCTCGGA ATTTCTCGAT TCTTTGAGGC TGAGATTGCT GATTGCTTAA GTCACATTCA TAGATATTGG AATGATAAGG GGCTTTTCAG TGGACGTGAA TCGGACTTTG TCGATATTGA CGACACATCC ATGGGTTTCA GACTTCTAAG AATGCAAGGC TATGATGTTA GTCCAAATGT ACTGAGGAAT TTCAAGAATG GTGACAAGTT TTCATGTTAC GGAGGTCAAA CGATCGAGTC ATCAACTCCA ATATACAATC TGTACAGACC TTCTCAATTC CGGTTTCCAG GAGAAGAAAT TCTTGAAGAA GCCGACAAGT TCGCCCATGA GTTCTTGTCC GAACAGCTTG GCAACAACCA ATTGCTTGAT AAATGGGTTA TATCCGACCG CTTGCAGGAA GAGATAAGTA TTGGATTGGG GATGCCATTT TATGCCACCC TTCCCAGAGT TGAAGCAAGC TACTATATAC AACATTACGC TGGTGCCGAC GACGTGTGGA TCGGCAAGAC ACTCTACAGG ATGCCGGAAA TAAGTAATGA TACATACCTG GAGCTAGCAA GAAATGATTT CAAGAGATGC CAAGCACAAC ATCAGTTCGA GTGGATCTAC ATGCAAGAAT GGTATGAGAG TTGCAACATT GAAGAATTCG GGATAAGCCG AAAGGAGCTC CTTCGCGTTT ACTTTTTGGC TTGCTCTAGC ATCTTTGAGG TCGAGAGGAC TAAAGAGAGA ATGGCATGGG CAAAATCTCA AATTATTTCT AGAATGATCA CTTCTTTCTT TAATAAACAA ACTACTTCAT CTGAGGAAAA AGAAACACTT TTAACCGAAT TCAGAAACAT CAACGGTCTG CACAAATCAA ACAATACAAG AGATGGAGAT ATGAACATTG TGCTTGCAAC CCTCCATCAA TTCTTCGCTG GATTTGACAG ATATACTAGC CATCAACTGA AAAATGCTTG GGGAGTATGG TTGACCAAGC TGCAACGAGG AGCAGTAGAC GGTGGAGCAG ACGCAGAGCT GATAACAACC ACCATAAACG TATGCGCCGG TCATATAGCT CTTAAGGAAG ACATATTGTC CCACGATGAG TACAAGACTC TCACCGACCT CACCAGCAAG ATTTGTCAGC AGCTTTCTCA TATTCAAAAC GAAAAGGTTG TGGAAATTGA CGGTGGGATT ACAGCAAAAT CTAGGTTGAA GAATGAGGAA CTGCAACGTG ACATGCAATC ATTGGTGAAA TTAGTACTTG AGAAATCAGT TGGGCTCAAC CGGAATATAA AGCAAACATT TCTAACGGTT GCAAAAACAT ACTACTACAG AGCCTACAAT GCTGAGGAAA CTATGGATGC CCATATATTC AAAGTTCTTT TCGAACCAGT TGCGTGA

Ajuga replans cleroda-4(18),13E-dienyl diphosphate synthase (ArTPS2) was identified and isolated as described herein. ArTPS2 was identified as a (5R,8R,9S,10R) neo-cleroda-4(18),13E-dienyl diphosphate [38] synthase. In addition, the combination of ArTPS2 and Ss-SS enzymes generated neo-cleroda-4(18),14-dien-13-ol [37]. These compounds are shown below.

ArTPS2 is of particular interest for applications in agricultural biotechnology, for example, because it is useful for production of neo-clerodane diterpenoids. Neo-clerodane diterpenoids, particularly those with an epoxide moiety at the 4(18) position, have garnered significant attention for their ability to deter insect herbivores (Coll et al., Phytochem Rev 7(1):25 (2008); Klein Gebbinck et al. Phytochemistry 61(7):737-770 (2002); Li et al. Nat Prod Rep 33(10):1166-1226 (2016)). The 4(18)-desaturated products produced by ArTPS2 (e.g., compounds 37 and 38 with the ═CH₂ 4(18) desaturation projecting from the A ring) the can be used in biosynthetic or semisynthetic routes to yield potent insect antifeedants.

The Ajuga reptans cleroda-4(18),13E-dienyl diphosphate synthase (ArTPS2) can have the amino acid sequence shown below (SEQ ID NO:23).

MSFASQATSL LSSPNRLGHV PTPSSPARFA AGGAPFWKIL FTARSNGQYK AISRARNQGN VEYIDEIQKG PQVVLEAENS LEDDTQKDTD QIRELVENVR VKLQNIGGGG ISISAYDTAW VALVEDINGS GQPQFPTSLD WISNHQFPDG SWGSSKFLYY DRILCTLACI VALKTWNVHP DKYHKGLDFI RENIHKLADE EEVHMPIGFE VAFPSIIETA KKVGIEIPED FPGEKEIYAK RDLKLKKIPM DILHKMPTPL LFSIEGMEGL DWQKLFKFRD DGSFLTSPSS TAYALQQTKD ELCLKYLTDL VKKDNGGVPN AFPVDLFDRN YTVDRLRRLG ISRYFQPEIE ECMKYVYRFW DKRGISWARN TNVQDLDDTA QGFRNLRMHG YEVTLDVFKQ FEKCGEFFSF HGQSSDAVLG MFNLYRASQV LFPGEHMLAD ARKYAANYLH KRRLNNRVVD KWIINKDLEG EVAYGLDVPF YASLPRLEAR FYIEQYGGSD DVWIGKALYR MVNVSCDTYL ELAKLDYNKC QSVHQNEWKS FQKWYKSCSL GEFGFSEGSL LQAYYIAAST IFEPEKSGER LAWAKTAALM ETIQQLSSQQ KREFVDEFKH KNILKNENGE RYRSSTSLVE TLISTVNQLS SDILLEQGRD VHQELCHVWL KWLSTWEERG NLVEAEAELL LRTLHLNSGL DESSFSHPKY QQLLEVSTKV CHLLRLFQKR KVYDPEGCTT DIATGTTFQI EACMQELVKL VFSRSSEDLD SLTKLRFLDV ARSFYYTABC DPQVVESHID KVLFEKVV A nucleic acid encoding the Ajuga reptans cleroda-4(18),13E-dienyl diphosphate synthase (ArTPS2) with SEQ ID NO:23 is shown below as SEQ ID NO:24.

ATGTCATTTG CTTCCCAAGC CACCTCCCTC CTATCATCCC CCAACCGTCT CGGCCATGTT CCGACGCCAA GCTCGCCGGC TCGTTTCGCT GCCGGTGGTG CCCCATTTTG GAAGATATTA TTTACAGCTA GGTCTAATGG GCAGTATAAA GCTATTTCAA GAGCTCGTAA CCAAGGAAAT GTAGAGTACA TTGATGAGAT TCAGAAAGGC CCGCAAGTCG TATTGGAGGC AGAAAACAGC TTGGAAGATG ACACACAAAA AGATACTGAT CAGATAAGGG AACTAGTGGA AAATGTCCGA GTAAAGCTGC AGAATATCCG TGGTGGAGGG ATAAGCATAT CGGCGTACGA CACCGCATGG GTGGCGCTGG TGGAGGACAT CAACGGCAGT GGCCAGCCAC AGTTTCCGAC GAGCCTCGAT TGGATATCGA ACCATCAGTT CCCTGATGGG TCATGGGGCA GCAGCAAGTT TTTGTATTAT GATCGGATTC TATGCACATT AGCATGTATA GTTGCATTGA AAACCTGGAA TGTGCATCCT GATAAGTACC ACAAAGGGTT GGATTTCATC AGAGAGAACA TTCACAAGCT TGCGGACGAA GAAGAAGTGC ACATGCCAAT TGGGTTCGAA GTGGCATTCC CATCAATTAT TGAAACAGCT AAAAAAGTAG GAATCGAAAT CCCTGAGGAT TTTCCTGGCA AGAAAGAAAT TTATGCAAAA AGAGATTTAA AGCTAAAAAA AATACCAATG GATATACTGC ATAAAATGCC CACACCATTG CTCTTCAGCA TAGAAGGAAT GGAAGGCCTT GACTGGCAAA AGCTATTCAA ATTCCGCGAT GATGGCTCGT TTCTTACGTC TCCGTCCTCA ACAGCCTATG CACTCCAGCA AACAAAGGAT GAGCTATGCC TCAAGTATCT AACAGATCTT GTCAAGAAAG ACAACGGAGG AGTTCCGAAT GCATTTCCAG TAGACCTGTT TGATCGTAAC TATACAGTAG ACCGCTTGCG AAGGCTAGGA ATTTCACGGT ACTTTCAACC TGAAATTGAA GAATGCATGA AATATGTTTA CAGATTTTGG GATAAAAGAG GAATTAGCTG GGCAAGAAAT ACCAATGTTC AGGACCTTGA TGACACTGCA CAGGGATTCA GGAATTTAAG GATGCATGGT TATGAAGTCA CTCTAGATGT TTTCAAACAA TTTGAGAAAT GTGGAGAGTT TTTCAGTTTT CATGGGCAAT CCAGCGATGC TGTTTTAGGA ATGTTCAACT TGTACCGGGC TTCTCAGGTT TTATTTCCGG GAGAACACAT GCTTGCAGAT GCGAGGAAGT ATGCAGCCAA CTATTTGCAT AAACGAAGAC TTAATAATAG GGTGGTCGAC AAATGGATTA TCAACAAAGA CCTTGAAGGC GAGGTGGCAT ATGGGCTAGA TGTTCCGTTC TACGGCAGCC TACCTCGACT CGAAGCAAGG TTCTACATAG AACAATATGG GGGTAGTGAT GATGTGTGGA TTGGAAAAGC TTTATACAGA ATGGTAAATG TAAGCTGCGA CACTTACCTT GAGCTAGCAA AATTAGACTA CAACAAATGC CAATCCGTGC ATCAGAATGA GTGGAAAAGC TTTCAAAAAT GGTACAAAAG TTGCAGTCTT GGGGAGTTTG GGTTCAGTGA AGGAAGCCTA CTCCAAGCTT ACTACATAGC AGCCTCAACT ATATTCGAGC CAGAGAAATC AGGAGAACGC CTAGCTTGGG CTAAAACAGC AGCTCTAATG GAGACAATTC AACAACTTTC CAGCCAGCAA AAACGTGAAT TTGTTGATGA ATTCAAACAT AAAAACATAC TGAAGAATGA AAATGGAGAA AGGTATAGAT CAAGTACCAG TTTGGTAGAG ACTCTGATAA GCACTGTAAA TCAGCTCTCA TCAGACATAC TATTGGAGCA AGGCAGAGAC GTTCATCAAG AATTATGTCA CGTGTGGCTA AAATGGCTGA GTACATGGGA GGAAAGAGGA AACCTGGTGG AAGCGGAAGC CGAGCTTCTT CTGCGAACCT TACATCTCAA CAGCGGATTG GATGAATCAT CATTTTCCCA CCCTAAATAT CAACAGCTCT TGGAGGTGTC TACCAAAGTT TGCCACCTCC TTCGCCTATT TCAGAAACGA AAGGTGTATG ATCCCGAAGG GTGTACAACC GACATAGCAA CAGGAACAAC GTTCCAGATA GAAGCATGCA TGCAAGAACT AGTGAAATTA GTGTTCAGCA GATCCTCAGA AGATTTAGAT TCTCTTACTA AGTTGAGATT TTTGGATGTT GCTAGAAGTT TCTATTACAC TGCCCATTGT GATCCACAGG TGGTCGAGTC CCACATCGAT AAAGTATTGT TTGAGAAGGT AGTCTAG

The Plectranthus barbatus (H-)-Copalyl diphosphate synthase (CfTPS16) was identified and isolated using the methods described herein, and this CfTPS116 protein can have the amino acid sequence shown below (SEQ ID NO:25).

MQASMSSLNL NNAPAVCSSR SQLSAKLHPP EYSTVGAWLN RGNKNQRLGY RIRPKQLSKL TECRVASADV SGEIGKVCQS VRTPEEVNKK IEESIKYVKE LLMTSGDGRI SVAPYDTAIV ALIKDLEGRD APEFPSCLEW IANNQKDDGS WGDDFFCIYD RIVNTIASVV ALKSWNVHPD KIERGVSYIK ENAHKLKGGN LEHMTSGFEF VVPGCFDRAK ALGIEGLPYD DPIIKEIYAT KERRLSKVPK DMIYKVPTTL LFSLEGLGME DLDWQKILKL QSGDGSFLTS PSSTAYAFMQ TGDEKCYKFL QNAVRNCNGG APHTYPVDVF ARLWAVDRLQ RLGISRFFQP EIKFCLDHIK NVWTKNGVFS GRDSEFVDID DTSMGIRLLK MHGYDVDPNA LKHFKQEDGR FSCYGGQMIE SASPIYNLYR AAQLRFPGEE ILEEATKFAY NFLQQKLANN QIQEKWVISE HLIDEIKMGL KMPWYATLPR VEASYYLQYY AASGDVWIGK TFYRMPEISN DTYKELALLD FNRCQAQHQF EWIYMQEWYQ SNNIKEFGIS KKELLLAYFL AAATIFEPER SQERIVWAKT QVVSKMITSF LSQENALSSX QKTALFIDFG HSINGLNQIT SVEKENGLAQ TVLATFGQLL EEYDRYTRHQ LKNAWSQWFM KLQQGDDNGG ADAELLANTL NICAGHIAFN EDILSHNEYT SLSSLTNKIC QRLSQIRDNK ILEIEDGSIK DKELEQEMQA LVKLVLEETG GIDRNIKQTF LSVFKMFYYR AYHDAEAIDX HIFKVMFEPV V A nucleic acid encoding the Plectranthus barbatus (+)-Copalyl diphosphate synthase (CfTPS16) with SEQ ID NO:25 is shown below as SEQ ID NO:26.

ATGCAGGCTT CTATCTCATC TCTGAACTTG AACAATGCAC CGGCCGTCTG CAGCAGCAGG TCACAGCTAT CCGCTAAACT TCACCCGCCG GAATATTCCA CCGTGGGTGC ATGGCTGAAT CGTGGCAACA AAAACCAGCG GTTGGGCTAC CGGATTCGTC CAAAGCAACT ATCAAAACTA ACTGAGTGTC GAGTAGCAAG TGCAGATGTG TCACAAGAGA TTGGAAAAGT CGGCCAATCT GTTCGGACTC CTGAAGAGGT AAATAAAAAG ATAGAGGAAT CCATCAAGTA CGTGAAGGAG CTGCTGATGA CGTCGGGCGA CGGGCGAATC AGTGTGGCGC CCTACGACAC GGCCATAGTT GCCCTTATCA AGGACTTGGA AGGGCGCGAT GCCCCGGAGT TTCCATCTTG CTTGGAGTGG ATTGCAAACA ATCAAAAAGA CGATGGTTCT TGGGGGGATG ACTTCTTCTG CATCTATGAT CGGATCGTTA ATACCATAGC ATCCGTCGTC GCCTTAAAAT CATGGAATGT GCACCCAGAC AAGATTGAGA GAGGAGTATC CTACATCAAG GAAAACGCGC ATAAACTAAA AGGTGGGAAT CTCGAACACA TGACATCAGG GTTCGAGTTC GTGGTTCCCG CGTGTTTTGA CAGAGCCAAA GCCTTGGGCA TCGAAGGCCT TCCCTATGAT GATCCCATCA TCAAGGAGAT TTATGCTACA AAAGAAAGGA CATTGAGCAA GGTACCGAAG GACATGATCT ACAAAGTTCC GACAACTCTA TTGTTTAGTT TAGAGGGACT GGGCATGGAG GATTTGGACT GGCAAAAGAT ACTGAAACTG CAGTCGGGCG ACGGCTCATT CCTCACCTCT CCGTCGTCCA CCGCCTACGC ATTCATGCAG ACCGGAGACG AAAAATGCTA CAAATTCCTC CAGAACGCCG TCAGAAATTG CAACGGCGGA GCGCCGCACA CTTATCCAGT CGACGTCTTT GCACGGCTCT GGGCGGTCGA CCGACTTCAG CGACTCGGAA TTTCTCGCTT CTTTCAGCCC GAGATCAAGT TTTGCCTAGA CCACATCAAA AATGTGTGGA CTAAGAACGG AGTTTTCAGT GGACGGGATT CAGAGTTTGT GGATATCGAC GACACATCCA TGGGCATCAG GCTTCTGAAA ATGCACGGAT ACGATGTCGA CCCAAATGCA CTGAAACATT TCAAGCAGGA GGATGGGAGG TTTTCATGCT ACGGTGGTCA AATGATCGAG TCTGCATCTC CGATTTACAA TCTCTACAGG GCTGCTCAGC TTCGTTTTCC AGGAGAAGAA ATTCTTGAAG AAGCCACTAA ATTTGCCTAC AACTTCCTGC AACAGAAGCT GGCCAACAAT CAAATTCAAG AAAAGTGGGT CATATCCGAG CACCTAATTG ATGAGATAAA AATGGGATTG AAGATGCCAT GGTACGCCAC CCTACCTAGA GTTGAGGCTT CATACTATCT CCAATATTAT GCAGCTTCTG GCGACGTATG GATTGGCAAG ACTTTTTACA GGATGCCAGA AATAAGTAAT GACACGTACA AAGAGCTTGC ACTATTGGAT TTCAACCGAT GCCAAGCACA ACATCAGTTC GAATGGATTT ACATGCAAGA GTGGTATCAA AGCAACAACA TTAAAGAATT TGGGATAAGC AAGAAAGAGC TTCTTCTTGC TTACTTCTTG GCTGCTGCAA CCATTTTTGA ACCCGAACGA TCGCAAGAGC GGATCGTGTG GGCTAAAACC CAAGTTGTTT CTAAGATGAT CACATCGTTT CTGTCTCAAG AAAACGCTTT GTCATCGGAN CAAAAGACTG CACTTTTCAT CGATTTTGGG CATAGTATCA ATGGCCTCAA TCAAATAACT AGTGTTGAGA AAGAGAATGG GCTTGCTCAG ACTGTCCTGG CAACCTTCGG ACAACTACTC GAGGAATTCG ACAGATACAC AAGGCATCAA CTGAAAAATG CTTGGAGCCA ATGGTTCATG AAACTGCAGC AAGGAGATGA CAATGGCGGG GCAGACGCAG AGCTCCTAGC AAACACATTG AACATCTGCG CTGGTCATAT TGCTTTTAAC GAAGACATAT TATCTCACAA CGAATACACC TCTCTCTCCT CCCTCACAAA CAAAATCTGT CAGCGGCTAA GTCAAATTCG AGATAATAAG ATACTGGAAA TTGAGGATGG GAGCATAAAA GATAAGGAAC TAGAACAGGA AATGCAGGCG CTGGTGAAGT TAGTCCTGGA AGAAACCGGT GGCATCGACA GGAACATCAA GCAAACATTT TTGTCAGTTT TCAAAATGTT TTACTACAGA GCCTACCACG ATGCTGAGGC TATCGATGNC CATATTTTCA AAGTAATGTT TGAACCAGTC GTATGA

Hyptis suaveolens labda-7,13E-dienyl diphosphate synthase (HsTPS1) was identified and isolated as described herein, and is a (55, 95, 105) labda-7,13E-dienyl diphosphate [21] synthase. When HsTPS1 was expressed in N. benthamiana, labda-7,13(16), 14-triene [22] was formed. The combination of HsTPS1 with OmTPS3 produced labda-7,12E,14-triene [24].

The Hyptis suaveolens labda-7,13E-dienyl diphosphate synthase (HsTPS1) can have the amino acid sequence shown below (SEQ ID NO:27).

MAYMISISNL NCSSLINTNL SAKIQLHQGL KGTWLKTSKR MCMDQQVHGK QIAKVIESRV TDKDVSTAQD FEVLKVNRVE DLISSIKSSL KTMEDGRISV SPYSTSWIAL IPSIDGRQTP QFPSSLEWIV KHQLSDGSWG DALFFCVYDR LVNTIACIIA LHTWKVHADK VKKGVSFVKE NIWKLEDANE VHMTSGFEVI FPILLRRARD MGIDGLPSDD TPVVRMISAA RDHKLKKIPR EVMHQVTTIL LYSLEGLEDL DWSRLFKLQS ADGSFLTSPS STAFAFMQTN NHNCLRFITS VVQTFNGGAP DNYPIDIFAR LWAVDRLQRL GISRFFEQEI NDCLSYVYRF WNANGVFSAG ATNFCDLDDT SMAFRLLRLH GYDVDPNVLR KFKEGDRFCC HSGEVAMSTS PTYALYRASQ IQFPGEEILD EAFSFTRDYL QDWLARDQVL DKWIVSKDLP DEIKVGLEVP WYASLPRVEA AYYMQRHYGG STDAWVAKTC YRMPDVSNDD YLELARLDFK RCQAQHQSEL SYMQRWYDSC NVEEFGISRK ELLVAYFVAA ATIFEPERAT ERIVWAKTEI VSKMIKAFFG EDSLDQKTML LKEFRNSINN GSHRFMKSEH RIVNILLQAL QELLHGSDDC RIGQLKNAWY EWLMKFEGGD EASLWGEGEL LVTTLNICTA HFLQHHDLLL NHDYITLSEL TNRICLKLSQ IQVGEMNEMR EDMQALTKLV IGESCIVNKN IKQTFLAVAK TFYYRAYFDA DTVDLHIFKV LFEPIV A nucleic acid encoding the Hyptis suaveolens labda-7,13E-dienyl diphosphate synthase (HsTPS1) with SEQ ID NO:27 is shown below as SEQ ID NO:28.

ATGGCGTATA TGATATCTAT TTCAAATCTC AACTGTTCCT CGCTACTAAA CACCAATCTT TCAGCAAAGA TTCAGCTGCA CCAAGGTCTC AAAGGAACAT GGCTAAAAAC CAGCAAACGC ATGTGCATGG ATCAACAGGT TCATGGCAAG CAGATAGCAA AAGTGATCGA GAGCCGAGTT ACTGATAAGG ATGTTTCCAC TGCTCAGGAC TTTGAAGTGT TAAAGGTCAA TAGAGTGGAG GATCTGATAT CAAGCATTAA GAGTTCATTG AAGACAATGG AAGATGGAAG AATAAGCGTG TCGCCCTACA GCACATCATG GATCGCACTC ATTCCAAGTA TTGATGGGCG CCAGACGCCC CAGTTTCCAT CTTCACTGGA GTCGATCGTG AAGCATCAGC TATCAGATGG TTCATGGGGT GATGCCCTTT TTTTCTGCGT TTATGATCGT CTCGTAAATA CGATTGCATG CATCATTGCC CTGCACACCT GGAAGGTTCA TGCAGACAAG GTTAAAAAAG GAGTAAGTTT TGTGAAGGAA AATATATGGA AACTTGAAGA CGCCAACGAG GTCCACATGA CTAGTGGTTT CGAAGTTATA TTTCCCATCC TTCTTCGAAG AGCACGAGAC ATGGGAATTG ATGGTCTTCC TTCTGATGAT ACTCCAGTTG TTAGGATGAT TTCTGCTGCT AGGGATCACA AATTGAAAAA GATTCCGAGG GAGGTGATGC ACCAAGTGAC AACAACTCTA TTATATAGTT TGGAAGGGTT GGAAGATTTA GACTGGTCAA GGCTTTTCAA ACTTCAGTCA GCTGATGGTT CATTCTTAAC TTCTCCATCT TCAACTGCCT TCGCATTCAT GCAAACTAAT AACCACAATT GCTTGAGATT CATCACTAGC GTTGTCCAAA CATTCAATGG AGGAGCTCCA GATAACTATC CAATCGACAT CTTTGCGAGA CTGTGGGCAG TTGACAGGTT ACAGCGGTTA GGGATTTCTC GTTTCTTCGA GCAGGAGATA AATGATTGCC TAAGCTATGT ATATAGATTT TGGAATGCAA ATGGAGTTTT CAGTGCAGGA GCCACTAATT TTTGTGATCT TGACGACACA TCCATGGCTT TCCGGCTACT ACGTTTGCAT GGATATGATG TCGACCCAAA TGTTCTGAGG AAATTCAAAG AGGGAGACAG ATTCTGTTGC CACAGTGGTG AAGTGGCGAT GTCGACATCG CCAACGTACG CTCTCTACAG AGCTTCCCAA ATTCAGTTTC CAGGAGAAGA AATTCTGGAT GAAGCCTTCA GCTTCACTCG CGACTATCTA CAGGACTGGT TAGCAAGAGA TCAAGTTCTT GATAAGTGGA TTGTATCCAA GGACCTTCCA GATGAGATTA AGGTAGGACT AGAGGTGCCA TGGTATGCCA GCCTGCCACG GGTAGAGGCT GCTTATTACA TGCAACGACA TTACGGCGGG TCTACTGATG CGTGGGTGGC CAAGACTTGT TACAGGATGC CTGATGTGAG CAACGATGAT TACCTGGAGC TTGCAAGATT GGATTTCAAG AGATGTCAAG CCCAACATCA GACTGAATTG AGTTACATGC AACGATGGTA TGACAGTTGC AATGTCGAAG AATTCGGAAT AAGCAGAAAA GAGTTGCTTG TAGCTTATTT TGTGGCTGCT GCAACTATTT TTGAACCTGA GAGAGCAACT GAGAGAATTG TGTGGGCAAA AACTGAAATA GTTTCTAAGA TGATCAAAGC ATTTTTTGGT GAAGACTCAT TAGACCAAAA AACTATGTTG TTAAAAGAAT TCAGAAACAG CATCAATAAT GGCTCCCACA GATTCATGAA GAGTGAGCAT AGAATCGTCA ACATTCTACT ACAAGCCTTG CAGGAGCTAT TACATGGATC TGATGATTGT CGTATTGGTC AACTCAAAAA TGCTTGGTAT GAGTGGCTGA TGAAATTCGA GGGAGGAGAT GAAGCAAGTT TGTGGGGAGA AGGAGAGCTT CTTGTCACCA CCTTAAACAT TTGCACAGCT CATTTCCTTC AACACCATGA TTTACTGTTG AATCATGACT ACATAACTCT TTCTGAGCTC ACAAACAAGA TCTGCCTCAA GCTTTCTCAG ATTCAGGTAG GAGAAATGAA TGAAATGAGA GAAGATATGC AGGCGTTGAC GAAATTAGTG ATTGGGGAAT CATGCATCGT CAACAAAAAC ATTAAGCAAA CATTTCTTGC AGTTGCAAAG ACTTTCTATT ACAGAGCCTA CTTCGATGCC GACACCGTTG ATCTCCATAT ATTTAAAGTT CTATTTGAGC CCATTGTCTG A

Leonotis leonurus peregrinol diphosphate synthase (LITPS1) was identified and isolated using the methods described herein. The LITPS1 enzyme was identified as a peregrinol diphosphate (PgPP) [5] synthase, where the peregrinol diphosphate (PgPP) [5] compound is shown below.

The Leonotis leonurus peregrinol diphosphate synthase (LITPS1) can have the amino acid sequence shown below (SEQ ID NO:29).

MASTASTLNL TINSTPFVST KTQAKVSLTA CLWMQDRSSS RHVSLKHKFC RNQQLKCRAS LDVQQVRDEV FSTAQSPESV DKKIEERKKW VKNLLSTMDD GRINWSAYDT AWISLIKEFE GRDATQFPST LMRIAENQLA DGSWGDPDYD CSYDRIINTL ACVVALTTWN AHPEHNKKGI KYIKENMYKL EETPVVLMTS AFEVVFPALL NRAKNLGIQD LPYDMPIVKE ICKIGDEKLA RIPKKMMEKE PTSLMYAAEG VENLDWEKLL KQRTPENGSF LSSPAATAVA FMHTKDENCL RYIMYLLDKF NGGAPNVYPI DLWSRLWATD RIQRLGISRF FKEEIKEILS YVYSYWTDIG VYCTRDSKYA DIDDTSMGFR LLRMHGFKMD PNVFKYFQKD DRFVCLGGQM NDSPTATYNL YRAAQYQFPG EKILEDARKF SQEFLQHCID TNNLLDKWVI SPRFPEELKF GMEMTWYSCL PRIEARYYVQ HYGATEDVWL GYTFFRMEEI SNENYKELAK LDFSKCQAQH QTEWIHMQEW YESSNAKEFG ISRKDLLFAY FLAAASIFET ERAKERILWA KSQIICKMVK SYLENQTASL EHKIAFLTGF GDNNNGLHTI NKGSGPVNNV MRTLQQLLGE FDGYISSQLE NAWAAWLTKL EQGEANDGEL LATTLNICSG RIVYNEDTLS NKEYKAFADL TNKICQNLAQ IQNKKGDEIK DPNEGEKDKE VEQGMQALAK LVFEESGLER SIKETFLAVV RTYHYGAYVA DEKIDVHMFK VLFEPVE A nucleic acid encoding the Leonotis leonurus peregrinol diphosphate synthase (LITPS1) with SEQ ID NO:29 is shown below as SEQ ID NO:30.

ATGGCCTCCA CTGCATCCAC TCTAAATTTG ACCATCAATA GTACACCATT TGTAAGCACC AAAACGCAAG CAAAGGTTTC CTTGCCCGCA TGTTTATGGA TGCAGGATAG AAGCAGCAGT AGACACGTGT CGTTAAAACA CAAATTCTGT CGAAATCAAC AACTTAAGTG TCGAGCAAGT CTGGATGTTC AGCAAGTACG TGATGAAGTT TTTTCCACTG CTCAATCCCC TGAATCGGTG GATAAAAAAA TAGAGGAACG TAAAAAATGG GTGAAGAATT TGTTGAGTAC AATGGACGAT GGACGAATAA ATTGGTCAGC CTATGACACG GCATGGATTT CACTTATTAA AGAATTTGAA GGACGAGATG CTCCCCAGTT TCCGTCGACT CTCATGCGCA TCGCGGAGAA CCAATTGGCC GACGGGTCAT GGGGCGATCC AGATTACGAC TGCTCCTATG ATCGGATAAT AAACACACTA GCGTGTGTTG TAGCCTTGAC AACATGGAAT GCTCATCCTG AACACAATAA AAAAGGAATA AAATACATCA AGGAAAATAT GTATAAACTA GAAGAGACGC CTGTTGTACT CATGACTAGT GCATTTGAAG TTGTGTTTCC GGCGCTTCTT AACAGAGCTA AAAACTTGGG CATTCAAGAT CTTCCCTATG ATATGCCCAT CGTGAAGGAG ATTTGTAAAA TAGGGGATGA GAAGTTGGCA AGGATACCAA AGAAAATGAT GGAGAAAGAG CCAACATCGC TGATGTATGC CGCGGAAGGA GTCGAAAACT TGGACTGGGA AAAGCTTCTG AAACAGCGGA CACCCGAGAA TGGCTCGTTC CTCTCTTCCC CGGCCGCAAC TGCCGTTCCA TTTATGCACA CAAAAGATGA AAATTGCTTA AGATACATCA TGTACCTTTT GGACAAATTT AATGGAGGAG CACCAAATGT TTATCCGATC GACCTCTGGT CAAGACTTTG GGCAACGGAC AGGATACAAC GTCTGGGAAT TTCCCGCTTC TTTAAGGAAG AGATTAAGGA AATCTTAAGT TATGTCTATA GCTATTGGAC AGACATTGGA GTCTATTGTA CACGAGATTC CAAATATGCT GACATTGACG ACACATCCAT GGGATTCAGG CTTCTGAGGA TGCACGGATT TAAAATGGAC CCAAATGTAT TTAAATACTT CCAGAAAGAC GACAGATTTG TTTGTCTAGG TGGTCAAATG AATGATTCTC CAACTGCAAC ATACAATCTT TACAGGGCTG CTCAATACCA ATTTCCAGGT GAAAAAATTC TAGAAGATGC TAGAAAGTTC TCTCAAGAGT TTCTACAACA TTGTATAGAC ACCAATAACC TTCTAGATAA ATGGGTGATA TCCCCGCGCT TTCCGGAAGA GTTGAAATTT GGAATGGAGA TGACATGGTA TTCCTGCCTA CCACGAATTG AGGCTAGATA CTACGTACAA CATTATGGTG CTACAGAGGA CGTCTGGCTT GGAAAGACTT TTTTCAGGAT GGAAGAAATC AGTAATGAGA ACTATAAGGA GCTTGCAAAA CTTGATTTCA GTAAATGCCA AGCACAACAT CAGACAGAGT GGATTCATAT GCAAGAGTGG TATGAAAGTA GCAATGCTAA GGAATTTGGG ATAAGCAGAA AAGACCTACT TTTTGCTTAC TTTTTGGCTG CAGCTTCCAT ATTTGAAACC GAAAGGGCAA AAGAGAGAAT TCTGTGGGCA AAATCTCAAA TTATTTGCAA GATGGTTAAG TCATATCTGG AAAACCAAAC GGCGTCGTTG GAGCACAAAA TCGCCTTTTT AACTGGATTC GGAGATAACA ACAATGGCCT GCACACAATT AATAAGGGGT CTGGACCTGT TAACAATGTC ATGAGAACCC TCCAACAGCT CCTTGGAGAA TTCGACGGAT ATATTAGTAG TCAATTGGAA AATGCTTGGG CAGCATGGTT GACGAAACTC GAGCAAGGCG AGGCCAACGA TGGCGAGCTC CTCGCAACCA CACTAAACAT TTGTTCTGGG CGTATTGTGT ATAACGAGGA TACATTATCG AACAAGGAGT ACAAGGCTTT CGCAGACCTC ACAAATAAAA TTTGTCAAAA TCTTGCTCAA ATCCAAAATA AAAAGGGTGA CGAAATTAAG GATCCGAATG AAGGCGAAAA GGACAAGGAA GTCGAGCAAG GCATGCAGGC ATTGGCTAAG TTAGTTTTTG AGGAATCTGG GCTTGAGAGG AGTATCAAAG AAACATTCTT AGCAGTGGTG AGAACTTATC ACTATGGGGC CTATGTTGCT GATGAGAAGA TTGATGTCCA CATGTTCAAG GTTTTGTTCG AACCAGTTGA ATGA

Nepeta mussinii (+)-copalyl diphosphate synthase (NmTPS1) was identified and isolated. The NmTPS1 enzyme can synthesize compound 31, 10 shown below.

The Nepeta mussinii (+)-copalyl diphosphate synthase (NmTPS1) can have the amino acid sequence shown below (SEQ ID NO:31).

MTSISSLNLS NAAAARRRLQ LPANVHLPEF HSVCAWLNSS SKHDPFSCRI HRKQKSKVTE CRVASVDASP VSDHKMSSPV QTQEEANKNM EESIEYIKNL LMTSGDGRIS VSAYDTSIVA LIKDIEGRDA PQFPSCLEWI GQNQKADGSW GDDFFCIYDR FVNTLACIVA LKSWNLHPHK IQKGVTYIKK NVHKLKDGRP ELMTSGFEIC VPAILQRAKD LGIQDLPYDD PMIKQITDTK ERRLKKIPKD FIYQLPTTLL FSLEGQENLD WEKILKLQSA DGSFLTSPSS TAAVFMHTKD EKCLKFIENA VKNCDGGVPH TYPVDVFARL WAVDRLQRLG ISRFFQPEIK YFLDHIQSVW TENGVFSGRD SQFCDIDDTS MGIRLLKMHG YKIDPNALEH FKQEDGKFSC YGGQMIESAS PIYNLYRAAQ LRFPGEEILE EAIKFSYNFL QEKLAKDEIQ EKWVISEHLI DEIKTGLKMP WYATLPRVEA AYYLDYYAGS GDVWIGKTFY RMPEISNDTY KEMAILDFNR CQAQHQFEWI YMQEWYESSN VKEFGISKKE LLVAYFLAAS TIFEPERAQE RIMWAKTKIV SKMIASSLNK QTTLSLDQKT ALFTQLEHSL NGLDSDEKDN GVAETKNLVA TFQQLLDGFD KYTRHQLKNA WSQWLKQVQQ GEATGGADAE LEANTLNICA GHIAFNEQVL SHNEYTTLST LTNKICHRLT QIQDKKTLEI IDGGIRYKEL EQEMQALVKL VVEENDGGGI DRNIKQTFLS VFKNYYYSAY HDAHTTDVHI FKVLFGPVV A nucleic acid encoding the Nepeta mussinii (+)-copalyl diphosphate synthase (NmTPS1) with SEQ ID NO:31 is shown below as SEQ ID NO:32.

ATGACTTCAA TATCCTCTCT AAATTTGAGC AATGCAGCAG CTGCTCGCCG CAGGTTACAA CTACCAGCAA ACGTTCACCT GCCGGAATTT CACTCCGTCT GTGCATGGCT GAATAGCAGC AGCAAACACG ATCCCTTTAG TTGCCGAATT CATCGAAAGC AAAAATCGAA AGTAACCGAG TGTCGAGTAG CAAGCGTGGA TGCATCACCA GTGAGTGATC ATAAAATGAG TTCTCCTGTT CAAACTCAAG AAGAGGCAAA TAAAAATATG GAGGAGTCAA TCGAGTACAT AAAGAATTTG TTGATGACAT CTGGAGACGG GCGAATAAGC GTGTCGGCAT ACGACACGTC AATAGTCGCC CTAATTAAGG ACATAGAAGG ACGCCACGCC CCGCAATTTC CATCATGCCT GGAGTGGATC GGGCAAAACC AAAAGGCCGA TGGCTCGTGG GGGGACGACT TCTTCTGTAT TTACGACCGC TTCGTAAATA CACTAGCATG TATCGTGGCC TTGAAATCAT GGAACCTTCA CCCTCACAAG ATTCAAAAAG GAGTGACATA CATCAAGAAA AACGTGCATA AGCTTAAAGA TGGGAGGCCT GAGCTGATGA CGTCAGGGTT CGAAATTTGT GTTCCCGCCA TTCTTCAAAG AGCCAAAGAC TTGGGCATCC AAGATCTTCC CTATGATGAT CCCATGATTA AACAGATCAC TGATACGAAA GAGCGACGAC TCAAAAAGAT ACCGAAGGAT TTTATATACC AATTGCCGAC GACTTTACTC TTCAGTTTGG AAGGGCAGGA GAATTTGGAC TGGGAAAAGA TACTCAAACT GCAGTCAGCT CACGGCTCCT TCCTTACTTC GCCGTCCTCC ACCGCCGCCG TCTTCATGCA TACCAAAGAT GAAAAATGCT TGAAGTTCAT AGAGAACGCC GTCAAAAATT GCGACGGCGG AGTGCCCCAT ACCTACCCAG TAGACGTGTT TGCAAGACTT TGGGCAGTTG ACAGACTACA ACGCCTAGGG ATTTCTCGCT TTTTTCAGCC TGAGATTAAA TATTTCTTAG ATCACATACA AAGCGTTTGG ACTGAGAACG GAGTTTTCAG TGGACGAGAT TCACAATTTT GCGACATTGA TGATACGTCC ATGGGGATAA GGCTTCTGAA AATGCATGGA TACAAAATCG ACCCAAATGC ACTTGAGCAT TTCAAGCAGG AGGATGGTAA ATTTTCGTGC TACGGTGGTC AAATGATCGA GTCTGCATCA CCGATATACA ATCTGTACCG AGCTGCTCAA CTCCGATTTC CAGGAGAAGA AATTCTTGAA GAGGCCATTA AATTTTCCTA TAACTTTTTG CAAGAAAAGC TAGCCAAGGA TGAAATTCAA GAAAAATGGG TCATATCGGA GCACTTAATT GATGAGATTA AGATCGGGCT AAAGATGCCA TGGTACGCCA CTCTACCCCG AGTTGAAGCT GCATATTACC TGGACTATTA TGCAGGATCC GGCGATGTGT GGATTGGCAA GACTTTCTAC AGGATGCCAG AAATCAGTAA TGATACATAC AAAGAAATGG CCATTTTGGA TTTCAACCGA TGCCAAGCAC AACATCAGTT TGAATGGATT TACATGCAAG AGTGGTATGA AAGTAGCAAC GTAAAGGAAT TTGGGATAAG CAAAAAAGAG CTACTTGTTG CTTATTTCTT GGCTGCATCA ACCATATTTG AACCGGAAAG AGCACAAGAG AGGATTATGT GGGCAAAAAC AAAAATTGTT TCCAAAATGA TCGCATCATC TCTTAACAAA CAAACCACTC TATCGTTAGA CCAAAAGACT GCACTTTTTA CCCAACTCGA ACATAGTCTC AATGGCCTCG ACAGTGATGA GAAAGATAAT GGAGTAGCTG AGACGAAAAA TCTAGTGGCA ACCTTCCAGC AGCTGCTAGA TGGATTCGAC AAATACACTC GCCATCAATT GAAAAATGCT TGGAGCCAGT GGTTGAAGCA AGTGCAGCAA GGAGAGGCGA CCGGGGGCGC AGACGCGGAG CTGGAAGCAA ACACGTTGAA CATCTGTGCC GGTCATATCG CATTCAACGA ACAAGTATTA TCGCACAACG AATACACAAC TCTCTCCACA CTCACAAACA AGATCTGCCA CCGGCTTACC CAAATTCAAG ACAAAAAGAC GCTTGAGATA ATCGACGGCG GCATAAGATA TAAGGAGCTG GAGCAGGAGA TGCAGGCGTT GGTGAAATTA GTTGTTGAAG AAAACGACGG CGGCGGCATA GACAGGAATA TTAAACAAAC ATTTTTATCA GTTTTCAAGA ATTATTACTA CAGTGCCTAC CACGATGCTC ACACAACCGA TGTTCATATT TTCAAAGTAT TATTTGGACC GGTCGTCTGA

Origanum majorana (+)-copalyl diphosphate synthase (OmTPS1) was 10 identified and isolated as describe herein. The OmTPS1 enzyme can synthesize compound 31. OmTPS1 can also synthesize palustradiene [29] (shown below), when combined with OmTPS5.

The Origanum majorana (+)-copalyl diphosphate synthase (OmTPS1) can have the amino acid sequence shown below (SEQ ID NO:33).

MTDVSSLRLS NAPAAGGRLP LPGKVHLPEF RTVCAWLNNG CKYEPLTCRI SRRKISECRV ASLNSSQLIE KVGSPAQSLE EANKKIEDSI EYIKNLLMTS GDGRISVSAY DTSLVALIKD VKGRDAPQFP SCLEWIAQNQ MADGSWGDEF FCIYDRIVNT LACLVALKSW NLHPDKIEKG VTYINENVHK LKDGSTEHMT SGFEIVVPAT LERAKVLGIQ GLPYDHPFIK EIINTKERRL SKIPKDLIYK LPTTLLFSLE GQGELDWEKI LKLQSSDGSF LTSPSSTASV FMRTKDEKCL KFIENAVKNC GGGAPHTYPV DVFARLWAVD RLQRLGISRF FQHEIKYFLD HINSVWTENG VFSGRDSQFC DIDDTSMGVR LLKMHGYNVD PNALKHFKQE DGNFSCYPGQ MIESASPIYN LYRAAQLRFP GEEILEEASR FAFNFLQEKI ANHEIQEKWV ISEHLIDEIK LGLKMPWYAT LPRVEAAYYL EYYAGSGDVW IGKTFYRMPE ISNDTYKEVA ILDFNTCQAQ HQFEWIYMQE WYESSKVKDF GISKKDLLVA YFLAASTIFE PERTQERIIW AKTLILSRMI TSFMNKQATL SSQQKNAILT QLGESVDGLD KIYSGEKDSG LAETLLATFQ QLLDGFDRYT RHQLKNAWGQ WLMKVQQGEA NGGADAELIA NTLNICAGLI AFNEDVLLHS EYTTLSSLTN KICQRLSQIE DEKTLEVIEG GIKDKELEED IQALVKLALE ENGGCGVDRR IKQSFLSVFK TFYYRAYHDA ETTDLHIFKV LFGPVM A nucleic acid encoding the Origanum majorana (+)-copalyl diphosphate synthase (OmTPS1) with SEQ ID NO:33 is shown below as SEQ ID NG:34.

ATGACCGATG TATCCTCTCT TCGTITGAGC AATGCACCAG CTGCCGGCGG CAGGTTGCCG CTGCCGGGAA AGGTTCACCT GCCTGAATTT CGCACCGTTT GTGCATGGTT GAACAATGGC TGCAAATACG AGCCCTTGAC TTGTCGAATT AGTCGACGGA AGATATCTGA ATGTCGAGTA GCAAGTCTGA ATTCGTCGCA AGTAATTGAA AAGGTCGGTT CTCCTGCTCA ATCTCTAGAA GAGGCAAACA AAAAGATCGA GGACTCCATC GAGTACATTA AGAATCTATT GATGACATCT GGCGACGGGC GGATAAGTGT GTCGGCTTAC GACACGTCGC TAGTCGCCCT AATAAAGGAC GTGAAAGGAC GAGATGCCCC TCAGTTCCCG TCGTGCCTGG AGTGGATAGC GCAAAACCAA ATGGCCGACG GGTCGTGGGG GGATGAGTTC TTCTGTATTT ACGACCGGAT CGTGAATACA TTAGCATGCC TCGTTGCCTT GAAATCATGG AACCTTCACC CCGACAAGAT CGAAAAAGGA GTGACGTACA TCAACGAAAA TGTGCACAAA CTGAAAGACG GGAGCACCGA GCACATGACG TCAGGGTTCG AAATCGTGGT CCCCGCCACT CTAGAAAGAG CCAAAGTCTT GGGCATCCAA GGCCTCCCTT ATGATCATCC CTTCATTAAG GAGATTATTA ATACTAAGGA GCGAAGATTA AGCAAAATAC CCAAGGATTT GATATACAAA CTGCCAACGA CGCTGCTGTT CAGTTTAGAA GGGCAGGGAG AATTAGATTG GGAAAAGATA CTGAAACTGC AGTCAAGCGA TGGCTCCTTC CTTACTTCGC CCTCGTCGAC CGCCTCCGTC TTCATGCGGA CGAAAGACGA GAAATGCCTC AAGTTCATTG AGAACGCCGT TAAGAATTGC GGCGGGGGAG CGCCGCATAC TTACCCAGTG GATGTGTTTG CAAGACTTTG GGCAGTTGAC AGACTACAGC GATTAGGGAT TTCTCGATTC TTCCAACACG AGATTAAATA CTTCTTAGAT CACATTAAGA GTGTATGGAC CGAGAATGGA GTTTTCAGTG GACGAGATTC ACAATTTTGT GATATCGACG ACACTTCTAT GGGAGTTAGG CTTCTAAAAA TGCATGGATA CAATGTTGAT CCAAATGCGC TCAAGCATTT CAAGCAGGAG GATGGCAAAT TCTCTTGCTA CCCTGGCCAA ATGATCGAGT CTGCATCTCC GATATACAAT CTCTACCGAG CCGCTCAACT CCGGTTCCCC GGAGAAGAAA TTCTCGAAGA AGCAAGTCGA TTCGCCTTCA ACTTTCTGCA GGAAAAGATA GCCAACCATG AAATTCAAGA AAAATGGGTC ATATCTGAGC ACTTAATTGA TGAGATAAAG TTGGGACTGA AGATGCCATG GTACGCGACT CTGCCCCGAG TTGAGGCCGC TTATTATCTA GAGTATTATG CTGGCTCAGG CGACGTATGG ATTGGAAAGA CTTTCTACCG GATGCCGGAA ATCAGTAACG ATACGTATAA AGAGGTGGCC ATTTTGGATT TCAACACATG CCAAGCTCAA CACCAGTTTG AATGGATTTA CATGCAAGAG TGGTACGAAA GTAGCAAGGT TAAAGATTTC GGGATAAGCA AAAAGGACCT ACTTGTTGCT TACTTTCTGG CGGCATCGAC TATATTTGAA CCCGAAAGAA CACAAGAGAG GATTATTTGG GCAAAAACCC TAATTCTTTC TAGGATGATC ACATCATTTC TCAACAAACA AGCTACACTT TCATCCCAAC AAAAGAATGC CATCTTAACA CAACTTGGAG AGAGTGTCGA TGGCCTCGAT AAAATATATA GTGGTGAGAA AGATTCTGGG CTGGCTGAGA CTCTGCTGGC TACCTTCCAG CAACTGCTCG ACGGATTCGA TAGATACACT CGCCATCAAC TGAGAAATGC TTGGGGGCAA TGGTTGATGA AAGTGCAGCA AGGAGAGGCC AACGGTGGCG CCGACGCTGA GCTCATAGCA AACACACTCA ATATCTGCGC CGGCCTTATC GCCTTCAACG AAGACGTATT GTTGCACAGC GAATACACGA CTCTCTCCTC CCTCACCAAC AAAATATGCC ACCGCCTTAG CCAGATTGAA GATGAAAAGA CGCTTGAAGT GATTGAAGGG GGCATAAAAG ATAAGGAACT GGAGGAGGAT ATTCAGGCGT TGGTGAAGCT AGCCCTCGAA GAAAACGGCG GCTGCGGCGT CGACAGAAGA ATCAAGCAGT CATTCTTATC AGTATTCAAG ACTTTTTACT ACAGAGCCTA CCATGATGCT GAGACCACCG ATCTTCATAT TTTCAAAGTA CTGTTGGGGC CGGGTATGTG A

A Perovskia atriplicifolia (+)-Copalyl diphosphate synthase (PaTPS1) enzyme was identified and isolated as described herein. This Perovskia atriplicifolia (+)-Copalyl diphosphate synthase (PaTPS1) enzyme was identified to be a (+)-copalyl diphosphate ((+)-CPP) synthase that can synthesize compound 31. The Perovskia atriplicifolia (+)-Copalyl diphosphate synthase (PaTPS1) can have the amino acid sequence shown below (SEQ ID NO:35).

MTSMSSLNLS RAPATTHRLQ LQAKVHVPEF YAVCAWLNSS SKQAPLSCQI RCKQLSRVTE CRVASLDASQ VSEKDTSHVQ TPDEVNKKIE DYIEYVKNLL MTSGDGRISV SPYDTSIVAL IKDSKGRNIP QFPSCLEWIA QHQMADGSWG DQFFCIYDRI LNTLACVVAL KSWNVHGDMI EKGVTYVKEN VHKLKDGNIE HMTSGFEIVV PALVQRAKDL GIQGLPYDDP LIKEIADTKE RRLKKIPKDM IYQTPTTLLF SLEGQGDLEW EKILKLQSGD GSFLTSPSST AHVFVQTKDE KCLKFIENAV KNCSGGAPHT YPVDVFARLW AIDRLQRLGI SRFFQPEIKY FIDHINSVWT ENGVFSGRDS EFCDIDDTSM GIRLLKMHGY KVDPNALNHF KQQDGKFSCY GGQMIESASP IYNLYRAAQL RFPGEEILEE ASKFAFNFLQ EKIANDQFQE KWVISDHLID EVKLGLKMPW YATLPRVEAA YYLQYYAGSG DVWIGKVFYR MPEISNDTYK ELAILDFNRC QAQHQFEWIY MQEWYHRSSV SEFG1SKKEL LRTYFLAAAT IFEPERTQER LVWAKTQIVS RMITSFVNNG TTLSLDQMTA LATQIGHNFD GLDQIISAMK DHGLAGTLLT TFQQLLDGFD RYTRHQLKNA WSQWFMKLQQ GEANGGEDAE LLANTLNICA GFIAFNEDVL SHDEYTTLST LTNKICKRLS QIQDKKALEV VDGSIKDKEL EQDMQALVKL VLEENGGGVD RNIKQTFLSV FKTFYYTAYH DDETTDVHIF KVLFGPVV A nucleic acid encoding the Perovskia atriplicifolia (+)-Copalyl diphosphate synthase (PaTPS1) enzyme with SEQ ID NO:35 is shown below as SEQ ID NO:36.

ATGACCTCTA TGTCCTCTCT AAATTTGAGC AGAGCACCAG CTACCACCCA CCGGTTACAG CTACAGGCAA AGGTTCACGT GCCGGAATTT TATGCCGTGT GTGCATGGCT GAATAGCAGC AGCAAACAGG CACCCTTGAG TTGCCAAATT CGCTGCAAGC AACTATCAAG AGTAACTGAA TGTCGGGTAG CAAGTCTGGA TGCGTCGCAA GTGAGTGAAA AAGACACTTC TCATGTCCAA ACTCCCGATG AGGTGAACAA AAAGATCGAG GACTATATCG AGTACGTCAA GAATCTGTTG ATGACGTCGG GCGACGGGCG AATAAGCGTG TCGCCCTACG ACACGTCAAT AGTCGCCCTT ATTAAGGACT CGAAAGGGCG CAACATCCCG CAGTTTCCGT CGTGCCTCGA GTGGATAGCG CAGCACCAAA TGGCGGATGG CTCATGGGGG GATCAATTCT TCTGCATTTA CGACCGGATT CTAAATACAT TAGCATGTGT CGTAGCTTTG AAATCCTGGA ACGTTCACGG TGACATGATC GAAAAAGGAG TGACGTACGT CAAGGAAAAT GTGCATAAGC TTAAAGATGG GAATATTGAG CACATGACGT CGGGGTTCGA AATTGTGGTT CCCGCCCTTG TTCAAAGAGC CAAAGACTTG GGCATCCAAG GCCTGCCCTA TGATGATCCC CTCATCAAGG AGATTGCTGA TACAAAAGAA AGAAGATTGA AAAAGATACC CAAGGATATG ATTTACCAAA CGCCAACGAC ATTACTATTC AGTTTAGAAG GGCAGGGAGA TTTGGAGTGG GAAAAGATAC TGAAACTGCA GTCAGGCGAT GGCTCCTTCC TCACTTCGCC GTCATCCACC GCCCACGTGT TCGTGCAGAC CAAAGATGAA AAATGCTTGA AATTCATCGA GAACGCCGTC AAGAATTGCA GTGGAGGAGC GCCGCATACT TATCCAGTCG ATGTCTTCGC AAGACTTTGG GCAATTGACA GACTACAACG CCTAGGAATT TCTCGTTTCT TCCAGCCGGA AATTAAGTAT TTCATAGACC ACATCAACAG CGTTTGGACA GAGAACGGAG TTTTCAGTGG GCGAGATTCG GAATTTTGCG ATATTGATGA CACGTCCATG GGCATCAGGC TTCTCAAAAT GCACGGATAC AAAGTCGACC CAAATGCACT CAATCATTTC AAGCAGCAAG ATGGTAAATT TTCTTGCTAC GGTGGTCAAA TGATCGAGTC TGCATCTCCA ATATACAATC TCTACAGGGC TGCTCAGCTA CGATTTCCAG GAGAAGAAAT TCTTGAAGAA GCCAGTAAAT TTGCCTTTAA CTTTTTGCAA GAAAAAATAG CCAACGATCA ATTTCAAGAA AAATGGGTGA TATCCGACCA CTTAATCGAT GAGGTGAAGC TCGGGCTGAA GATGCCATGG TACGCCACTC TACCCCGGGT TGAGGCTGCA TATTATCTAC AATACTATGC TGGTTCTGGC GACGTATGGA TTGGCAAGGT TTTCTACAGG ATGCCGGAAA TCAGCAATGA TACATACAAA GAGCTGGCCA TATTGCATTT CAACAGATGC CAAGCACAGC ATCAGTTCGA ATGGATTTAT ATGCAAGAGT GGTATCACAG AAGCAGCGTT AGTGAATTCG GGATAAGCAA AAAAGAGCTG CTTCGTACTT ACTTTCTGGC TGCAGCAACC ATATTCGAAC CCGAGAGAAC ACAAGAGAGG CTTGTGTGGG CAAAAACCCA AATTGTCTCT AGGATGATCA CATCATTTGT TAACAATGGA ACTACACTAT CTTTGGACCA AATGACTGCA CTTGCAACAC AAATCGGCCA TAATTTCGAT GGCCTCGATC AAATAATTAG TGGTATGAAA GATCATGGAC TGGCTGGGAC TCTGCTGACA ACCTTCCAGC AACTTCTAGA TGGATTCGAC AGATACACTC GCCATCAACT CAAAAATGCT TGGAGCCAAT GGTTCATGAA ACTCCACCAA GGGGAGGCGA ACGGCGGGGA AGACGCGGAG CTCCTAGCAA ACACGCTCAA CATCTGCGCG GGTTTCATTG CTTTCAACGA AGACGTATTG TCGCACGATG AATACACGAC TCTCTCCACC CTTACAAACA AAATCTGCAA GCGCCTTAGC CAAATTCAAG ATAAAAAGGC GCTGGAAGTT GTCGACGGGA GCATAAAGGA TAAGGAGCTC GAACAGGATA TGCAGGCGTT GGTGAAGTTG GTCCTTGAAG AAAATGGCGG CGGCGTCGAC AGGAACATCA AACAGACATT TTTGTCCGTT TTCAAGACTT TTTACTACAC CGCCTACCAC GATGATGAGA CCACTGATGT TCATATTTTC AAAGTACTGT TTGGACCGGT CGTATGA

Pogostemon cablin (10R)-labda-8,13E-dienyl diphosphate synthase (PcTPS1) was identified and isolated as described herein. This Pogostemon cablin (10R)-labda-8,13E-dienyl diphosphate synthase (PcTPS1) enzyme was identified to be a (10R)-labda-8,13E-dienyl diphosphate synthase, which can synthesize compound 25.

The combination of PcTPS1 and SsSS, both in-vitro, and in N. benthamiana expression produced (10R)-labda-8,14-en-13-ol [26], shown below.

This Pogostemon cablin (10R)-labda-8,13E-dienyl diphosphate synthase (PcTPS1) can have the amino acid sequence shown below (SEQ ID NO:37).

MSFASQSHVA FVLRRPSAVA PPPPTRIPTT AALSPLKPGD FSHGRSSFMP TSIKCNAIST SRVEEYKYTD DHNQSGLLEH DGLISDKINE LVTKIQLMLQ NMDDGEISIS PYDTAWVSLV EDVGGNDRPQ FPTSLEWISN NQLPDGSWGD PNAFLVHDRI LNTLACVVAL KSWKMHPHKC NRGVSFVREN IYRMDDEKEE HMPNGFEVVF PALLQKAKTL NIDIPYEFPG IQKFYAKRDL KFARIPMDIL HSVPTTLLFS LEGVRCGLDL DWGKLLELQA ADGSFLYSPS STAFALEQTK DQNCLKYLSK LVRKFDGGVP NVYPVDLFEH NWAVDRLQRL GISRYFTPEI NQCLDYSYRY WSNSKGMYSA SNSQIQDVDD TAMGFRLLRL NGYDVSTQGF RQFEAGGDFF CFAGQSSQAV TGMYNLYRAS QVMFPGEKLL EDAKKFSTNF LQQKRANNQL TDKWVIAKDV PAEVGYALDI PWYASLPRLE ARFFIQQYGG DDDVWIGKTL YRMGYVNNNT YLELAKLDYN TCQRLHQHEW ITIQRWYEIN LKITSVGLSK RGVLLSYYLA AANLFEPQNS THRIAWAKTS ILVSAIQLSP LQKRDFINQF HRSTANNGYE TSNVLVKSVI KGVHETSMDA MLTHNKDIHR QLFNAWRKWM SVWEEGGDGE AELLLSTLNT CDGVDESTFS DPKYEHLLEI TVRVTHQLHL IQNAETKRVG DREEIDLSMQ QLVKLVFTKS SSDLDSCIKQ RFFAIARSFY YVAHCDPEMV DSHIAKVLFE RVM A nucleic acid encoding the Pogostemon cablin (10R)-labda-8,13E-dienyl diphosphate synthase (PcTPS1) enzyme with SEQ ID NO:35 is shown below as SEQ ID NO:38.

ATGTCATTTG CTTCTCAATC ACATGTCGCC TTTGTACTCC GACGGCCATC TGCCGTTGCT CCGCCACCAC CGACTAGAAT TCCGACAACA GCCGCTCTTT CTCCTCTCAA ACCAGGTGAT TTTTCCCATG GCAGATCATC ATTTATGCCC ACTTCCATTA AATGTAATGC AATTTCCACA TCTCGCGTCG AAGAATACAA GTACACGGAT GATCATAATC AGAGTGGTTT ATTGGAGCAT GATGGTTTGA TATCAGACAA GATAAATGAA TTGGTGACCA AGATACAATT GATGCTACAA AACATGGATG ACGGAGAGAT AAGCATCTCC CCATATGACA CCGCATGGGT GTCGTTGGTG GAGGATGTGG GCGGCAACGA CCGCCCACAG TTTCCTACGA GCCTGGAGTG GATATCGAAT AACCAGCTCC CCGACGGCTC GTGGGGCGAC CCGAATGCCT TTTTGGTGCA CGACCGTATC CTCAACACAT TGGCATGCGT CGTTGCACTC AAATCCTGGA AAATGGACCC CCACAAATGC AATAGAGGAG TTAGTTTCGT CACAGAAAAT ATATACAGAA TGGATGATGA AAAAGAGGAA CACATGCCAA ATGGATTCGA AGTGGTATTT CCAGCACTCC TTCAAAAAGC GAAAACCCTA AACATTGATA TCCCGTACGA GTTTCCAGGA ATACAAAAAT TTTATGCCAA AAGAGATTTA AAATTCGCCA GGATTCCAAT GGATATATTG CATAGCGTTC CGACAACATT ACTGTTCAGC TTAGAAGGTG TAAGATGTGG TCTTGATCTG GATTGGGGGA AGCTTCTAGA ATTGCAAGCT GCTGATGGCT CATTTCTCTA CTCTCCATCC TCTACTGCCT TTGCACTAGA ACAAACCAAG GATCAAAACT GCCTCAAATA TCTATCTAAA CTTGTTCGAA AATTCGATGG CGGAGTACCC AACGTGTACC CGGTGGACTT GTTCGAACAT AATTGGGCAG TTGATCGTCT CCAAAGGCTC GGAATTTCTC GTTATTTTAC GCCTGAAATC AACCAATGTC TTGATTATTC TTACAGATAT TGGTCAAATA GTAAAGGGAT GTACTCGGCA AGCAATTCCC AGATTCAGCA CGTTGATGAC ACCGCCATGG GATTCAGGCT TTTGAGACTC AACGGCTACG ATGTCTCTAC ACAAGGGTTT AGGCAATTCG AGGCAGGGGG GGACTTCTTC TGCTTCGCGG GGCAGTCGAG CCAAGGTGTA ACCGGAATGT ACAACCTCTA CAGAGCTTCC CAAGTGATGT TCCCTGGAGA GAAGCTACTG GAAGATGCCA AGAAATTCTC CACCAACTTC TTGCAACAAA AACGAGCCAA TAACCAGCTC ACTGACAAGT GGGTTATTGC CAAAGATGTT CCAGCTGAGG TGGGATATGC CTTGGATATT CCCTGGTATG CCAGTCTGCC CCGACTGGAA GCAAGATTTT TCATACAACA ATACGGTGGA GACGACGACG TTTGGATCGG CAAAACCTTG TATAGAATGG GATATGTGAA CAACAACACT TATCTGGAAC TCGCAAAGCT AGACTACAAC ACCTGCCAAA GGTTGCATCA GCATGAGTGG ATAACCATTC AACGATGGTA CGAAATTAAT TTAAAAATTA CTAGTGTTGG GTTGAGCAAA AGAGGGGTCC TGTTGAGTTA TTACTTAGCC GCAGCCAATC TGTTTGAGCC TCAAAACTCA ACACACCGCA TCGCTTGGGC CAAAACTTCG ATTTTAGTAA GCGCTATTCA ACTTTCTCCC CTCCAAAAGC GCGACTTTAT TAACCAATTC CACCGCTCCA CCGCAAATAA TGGGTATGAA ACAAGTAATG TGTTGGTGAA GAGTGTAATC AAGGGTGTGC ATGAGCTCTC CATGGACGCT ATGTTGACGC ACAATAAAGA CATACATCGC CAACTTTTTA ATGCTTGGCG AAAGTGGATG TCAGTGTGGG AAGAGGGAGG TGATGGAGAA GCGGAGCTGT TATTGTCGAC GCTTAAGACG TGCGACGGAG TAGATGAATC CACATTCAGC GATCCCAAAT ACGAGCACCT CTTAGAGATC ACCGTCAGAG TCACCCACCA GCTTCATCTC ATTCAGAATG CAGAGACGAA GCGTGTGGGT GACCGTGAGG AAATAGATTT GAGCATGCAA CAACTTGTTA AGTTGGTGTT CACTAAATCA TCATCGGATC TGGATTCTTG TATCAAGCAA AGATTTTTTG CGATTGCCAG AAGTTTCTAT TACGTGGCTC ATTGTGATCC GGAGATGGTG GACTCCCACA TAGCCAAAGT ATTGTTTGAG AGGGTGATGT AG

Prunella vulgaris 11-hydroxy vulgarisane synthase (PvHVS) was identified and isolated as described herein. The Prunella vulgaris 11-hydroxy vulgarisane synthase (PvHVS) enzyme catalyzes the first committed step and forms the scaffold found in all Vulgarisms, a class of diterpenes with pharmaceutical applications (e.g., gout, cancer). For example, PvHVS can synthesize 11-hydroxy vulgarisane (shown below).

An example of a formula for several Vulgarism diterpenes is shown below.

Vulgarisms B (1) and C (2) exhibit modest cytotoxicity activity against human lung carcinoma A549 cell line (Lou et al. Tetrahedron Letters 58: 401-404 (2017)).

The Prunella vulgaris 11-hydroxy vulgarisane synthase (PvHVS) can have the amino acid sequence shown below (SEQ ID NO:39).

MSSLSIPFSS AICTSSIPKI STGHHRRTAR MPAHDTSRLV FRPSAVMVEG SPMTTSSNGK EVQRLITTEK PSMWKDIFST FSFDNQVQEK YLKEIEELKK EVRSTLMSAT HRKLFDLIDN LERMGIAYHF ETEIEDKLKQ AHASLEEEDD YDLFTTALRF RLLRQHRYHV SCDPFAKFVD QDNKLKESLS SDVEGLLSLF EASHLRIHNE DVLDEAlVFT THHLNRMKPQ LESPLKEEVK HALRYPLHKC LGILSLRFHI DRYENDKSRD EVVLRLGQVN FNYMQNIYMN ELYEITTWWN KLQMTSKVPY FRDRLVECYM WGLAYHFEPE YAPVRVLITK YYMTATTVDD TYDNYATLEE IELFTQAIDR WSEDEIDQLP DEYLKIVYKG LMNFTEEFRR DAEERGKGYV IPYFIEETKR ATQGYANEQR WIMKREMPSF EEYMVNSRVT SLMYVTYVAV VAVIESATKE TVDWALSDSD IFVYTNDIGR LIDDLATHRR ERKDGTMLTS MDYYMKEYGG TMEEGEAAFR KLMEEKWKLL NAAWVDTING KESKEIVVQV LDLARICGTL YGDEEDGFTY PEKNFAPLVA ALLMNPIHI A nucleic acid encoding the Prunella vulgaris 11-hydroxy vulgarisane synthase (PvHVS) enzyme with SEQ ID NO:39 is shown below as SEQ ID NO:40.

ATGAGCTCTC TCTCAATTCC CTTTTCTTCC GCCATTTGCA CTTCATCAAT CCCAAAGATC AGTACTGGGC ATCATCGCCG CACCGCGAGG ATGCCCGCGC ACGACACATC GCGTCTCGTC TTTCGCCCTT CAGCTGTGAT GGTGGAAGGA AGTCCGATGA CTACTTCAAG CAACGGGAAG GAAGTCCAAC GACTTATAAC CACTTTCAAG CCTAGCATGT GGAAAGATAT TTTTTCTACC TTCTCTTTCG ATAATCAGGT GCAAGAAAAG TATTTGAAAG AAATTGAGGA ATTGAAGAAA GAAGTAAGAA GCACACTAAT GAGTGCTACG CATAGGAAAT TGTTTGACTT GATCGACAAT CTCGAGCGTA TGGGAATCGC CTATCATTTC GAGACAGAAA TCGAAGACAA GCTCAAACAA GCTCATGCTT CTCTAGAGGA GGAAGATGAC TACGACTTGT TCACTACTGC ACTTCGCTTT CGTCTGCTCA GACAACATCG CTATCATGTT TCTTGCGATC CCTTTGCGAA ATTTGTTGAC CAAGACAACA AATTGAAAGA GAGTCTTAGT AGCGACGTCG AGGGGCTATT AAGCTTGTTC GAGGCATCCC ATCTTCGGAT CCACAACGAG GATGTTCTAG ATGAAGCTAT AGTGTTCACA ACCCATCACT TGAATCGAAT GATGCCACAA TTGGAATCGC CCCTTAAAGA AGAAGTGAAG CATGCTCTTC GATACCCCCT TCACAAGTGT CTTGGAATCC TTAGCCTTCG TTTTCATATC GACAGATATG AGAATGATAA GTCGAGGGAT GAAGTTGTTC TCAGACTAGG CCAAGTTAAT TTCAATTACA TGCAGAACAT TTACATGAAC GAGCTCTATG AAATCACCAC GTGGTGGAAC AAGTTGCAGA TGACTTCAAA AGTACCTTAC TTTAGAGATA GATTGGTAGA GTGCTATATG TGGGGTTTGG CATATCATTT CGAACCAGAA TACGCTCCCG TTCGAGTCCT CATTACCAAG TACTATATGA CCGCCACAAC TGTCGACGAT ACCTATGATA ATTATGCTAC ACTCGAAGAA ATCGAACTCT TCACTCAGGC CATTGACAGG TGGAGCGAGG ATGAGATTGA TCAGCTACCT GATGAATACC TAAAAATAGT GTACAAAGGT CTAATGAACT TCACTGAAGA GTTTAGACGT GACGCAGAAG AGCGAGCGAA AGGCTATGTG ATTCCTTACT TTATTGAAGA AACGAAGAGA GCAACACAGG GTTATGCAAA CGAGCAGAGG TGGATAATGA AGAGAGAAAT GCCGAGTTTT GAAGAGTATA TGGTGAACTC AAGGGTAACA TCACTTATGT ATGTGACCTA CGTTGCTGTT GTGGCAGTCA TAGAATCAGC TACCAAAGAA ACCGTAGATT GGGCGCTAAG TGACTCCGAT ATCTTTGTCT ACACTAACGA TATCGGCCGA CTTATCGACG ACCTTGCCAC TCATCGACGC GAGAGGAAAG ACGGGACAAT GCTTACATCG ATGGATTATT ACATGAAGGA ATATGGCGGT ACGATGGAAG AGGGGGAAGC TGCATTTAGG AAATTGATGG AGGAGAAATG GAAACTTTTG AATGCAGCAT GGGTAGATAC TATTAATGGA AAAGAGTCGA AGGAAATAGT TGTGCAAGTT CTCGACCTCG CCAGGATATG CGGAACGCTC TATCGGGACG AAGAAGATGG CTTCACCTAC CCAGAGAAGA ATTTTGCACC ACTCGTTGCT GCTCTATTGA TGAATCCTAT ACATATTTGA

A Chiococca alba ent-CPP synthase (CaTPS1) was identified and isolated. This CaTPS1 enzyme was identified that converts GGPP to ent-CPP [16].

The Chiococca alba ent-CPP synthase (CaTPS1) has the amino acid sequence shown below (SEQ ID NO:41).

1 MSSSTSAAAT LLGLSPASRR FVSFPPANGP IETITGIWSP 41 GKALHHFNFR LRCSTVSSPR TQELGQVSQN GMSGIKWHDI 81 VEEGVTEKGT LEANTSSWIK ESIEAIRWML RTMDDGDISI 121 SAYDTAWVAL VEDINGSGGP QFPSSLEWIA NNQLPDGSWG 161 DSDIFSAHDR ILNTLGCVVA LKSWNMHPEK SEKGLLYLRD 201 NIHKLEDENV EHMPIGFEVA FPSLIEIAKK LSIDIPDDSA 241 ILQEIYARRN LKLTRIPKDI MHTVPTTLLH SLEGMPELDW 281 KRLISLKCED GSFLFSPSST AFALTQTKDA DCLRYLIKTV 321 QKENGGVPNV YPVDLFEHIW AVDRLQRLGI SRYFQSEIRE 361 CIDYVHRYWT DKGICWARNT HVYDIDDTAM GFRLLRLHGY 401 DVSADVFRYY EKDGEFVCFA GQSNQAVTGM YNLYRASQVM 441 FPGENILSDA ERFSSEFLHD KRANNELLDK WIITKDLPGE 481 VAYALDVPWY ASLPRLETRL YLEQYGGEDD VWIGKTLYRM 521 QKVNNNIYLE LGKLDYNNCQ ALHQLEWRSI QKWYNECGLG 561 EYGLSERSLL LSYYLAAASI FEPERSKERL AWAKTTMLIR 601 TIESYLSSEQ MVEDHNGAFV SEFQYYCSNL DYVNGGRHKP 641 TQRLVRTLLG TLNQISLDAV LVHGRDIHQY LRQAWEKWLI 681 ALQEGDDSDM GQEEAELLVR TLNLCAGRYA SEELLLSHPK 721 YQQLLHITTR VCNQIRHFQH KKVQDGENGR ANMGDGITSI 761 SSIESDMQEL TKLVVGNTQN DLDADTKQTF LTVAKSFYYT 801 AHCNPGTINC HIAKVLFERV L

A nucleic acid encoding the Chiococca alba ent-CPP synthase (CaTPS1) with SEQ ID NO:41 is shown below as SEQ ID NO:42.

1 ATGTCTTCTT CTACCTCAGC AGCAGCAACC CTTCTCGGAT 41 TATCGCCGGC AAGCCGCCGG TTTGTATCAT TTCCTCCGGC 81 AAATGGACCT ATAGAAACTA TTACCGGTAT TTGGTCGCCC 121 GGCAAAGCTC TTCATCACTT TAATTTCCGT CTGCGTTGTA 161 GCACGGTGTC CAGTCCTCGC ACCCAAGAAT TGGGCCAGGT 201 GTCACAAAAT GGCATGTCTG GTATAAAGTG GCATGACATA 241 GTGGAAGAAG GAGTCACAGA AAAAGGAACT CTTGAGGCGA 281 ACACATCAAG CTGGATAAAA GAAAGCATAG AAGCCATTCG 321 TTGGATGCTG CGTACCATGG ATGACGGGGA TATCAGCATA 361 TCTGCTTATG ATACTGCATG GGTTGCCCTT GTGGAAGATA 401 TCAACGGAAG TGGCGGTCCT CAATTTCCTT CAAGCCTCGA 441 GTGGATTGCC AACAATCAGC TTCCTGATGG TTCATGGGGC 481 GACAGCGACA TCTTTTCAGC TCACGATCCG ATTCTCAACA 521 CTTTGGGATG CGTTGTTGCA TTAAAATCTT GGAACATGCA 561 CCCTGAAAAG AGTGAAAAAG GATTATTATA TTTAAGGGAT 601 AACATTCACA AGCTTGAGGA TGAAAATGTC GAGCACATGC 641 CTATCGGTTT TGAAGTGGCA TTTCCTTCAC TAATTGAGAT 681 AGCCAAAAAG TTGAGCATTG ATATTCCGGA TGATTCTGCA 721 ATCTTGCAGG AGATATATGC CAGAAGAAAT CTAAAGCTAA 761 CAAGGATACC GAAGGACATT ATGCACACAG TGCCCACAAC 801 ATTGCTCCAC AGCTTGGAAG GCATGCCAGA ACTAGACTGG 841 AAAAGGCTAA TATCTCTAAA GTGTCAGGAT GGTTCCTTTC 881 TGTTTTCTCC ATCCTCCACT GCTTTTGCCC TCACGCAAAC 921 TAAAGATGCT GATTGCCTCA GATATTTAAC TAAAACCGTA 961 CAAAAATTCA ATGGAGGAGT TCCCAATGTT TACCCCGTGG 1001 ACTTATTCGA ACACATCTGG GCTGTTGATC GACTTCAAAG 1041 ACTAGGAATT TCTCGATACT TCCAGTCAGA AATCCGCGAG 1081 TGCATCGATT ATGTTCACCG ATATTGGACG GATAAAGGTA 1121 TCTGTTGGGC TAGAAATACC CACGTTTATG ACATTGATGA 1161 TACAGCTATG GGTTTTAGAC TTCTAAGGTT GCATGGCTAC 1201 GATGTTTCTG CAGATGTTTT CAGATACTAT GAGAAGGATG 1241 GCGAATTCGT TTGCTTTGCC GGACAGTCAA ACCAGGCGGT 1281 GACCGGAATG TATAACCTGT ATAGAGCTTC TCAAGTGATG 1321 TTTCCAGGGG AGAATATACT TTCGGATGCT AGGAAATTCT 1361 CGTCCGAATT CTTGCATGAT AAGCGAGCCA ACAATGAGCT 1401 CCTAGATAAA TGGATCATAA CCAAAGATTT GCCTGGGGAG 1441 GTAGCATATG CTTTAGATGT TCCATGGTAT GCCAGTTTAC 1481 CTCGTTTAGA AACCAGATTG TATTTGGAAC AATATGGCGG 1521 CGAAGATGAT GTCTGGATTG GCAAGACATT GTACAGGATG 1561 CAAAAAGTTA ACAACAACAT CTATCTTGAA CTTGGCAAAT 1601 TAGATTACAA CAACTGTCAG GCATTGCATC AGCTTGAGTG 1641 GAGAAGCATC CAAAAATGGT ACAATGAATG CGGTCTTGGA 1681 GAGTACGGAT TAAGCGAGAG AAGCCTCCTT CTTTCGTATT 1721 ATTTGGCCGC AGCCAGTATA TTTGAAGCGG AGAGGTCAAA 1761 GGAACGGCTT GCCTGGGCCA AAACTACTAT GCTAATCCGC 1801 ACAATTGAAT CTTATTTGAG TAGTGAACAA ATGGTTGAGG 1841 ATCACAATGG AGCCTTTGTT AGCGAGTTCC AATACTATTG 1881 CAGTAACCTT GACTACGTAA ATGGTGGAAG GCATAAGCCA 1921 ACACAAAGGC TAGTGAGGAC TCTACTCGGA ACTTTAAATC 1961 AGATTTCTTT GGACGCAGTG TTAGTCCACG GCAGAGATAT 2001 CCATCAATAT TTGCGTCAAG CCTGGGAAAA GTGGTTGATA 2041 GCTTTGCAAG AGGGAGATGA TAGTGACATG GGTCAAGAGG 2081 AAGCAGAACT TTTAGTGCGC ACACTAAACC TATGCGCCGG 2121 TCGCTACGCA TCGGAGGAGC TATTGTTGTC CCATCCCAAG 2161 TATCAACAAC TTTTGCACAT CACTACTAGA GTCTGTAACC 2201 AAATTCGTCA TTTCCAACAC AAAAAGGTGC AAGATGGGGA 2241 AAATGGAAGA GCAAACATGG GTGATGGCAT CACAAGCATC 2281 AGCTCAATAG AGTCGGACAT GCAAGAACTA AGGAAATTAG 2321 TTGTCGGCAA TACCCAAAAC GATCTAGATG CTGATACGAA 2361 GCAAACATTT CTCACGGTGG CAAAAAGCTT CTACTACACC 2401 GCCCACTGCA ATCCCGGAAC AATCAATTGC CATATTGCTA 2441 AAGTATTATT TGAGAGAGTA CTTTGA

A Chiococca alba (5R,8S,9S,10S)-labda-13-en-8-ol diphosphate (ent-8-LPP) synthase (CaTPS2) was identified and isolated as described herein. This CaTPS2 enzyme was identified as an 5R,8S,9S,10S)-labda-13-en-8-ol diphosphate (ent-8-LPP) synthase, which converts GGPP to 5R,8S,9S,10S)-labda-8-en-8-oi diphosphate (ent-8-LPP, [7]).

The Chiococca alba (5R,8S,9S,10S)-labda-13-en-8-ol diphosphate (ent-8-LPP) synthase (CaTPS2) has the amino acid sequence shown below (SEQ ID NO:43).

1 MPVIKSHEFI EEVGPEKGTL KLSRSSRINE LVESIQTMLQ 41 SMDDGEISMS AYDTAWVALV EDINGSSYPQ FPMSLEWIAN 81 NQLPDGSWGD GSIFSVHDRI ISTLCCVLAL KSWNMHPDKS 121 EKGLLFIRDN IHKVGDESAE HMPIGFEVVF PSLIERAKNL 161 DIDIPDISAI LQEIYARRNL KLARIPKDIL YTVPTTLLHS 201 LEGMPELDWQ KLLPLKCEDG SFLFSPSCTA FALMQTKDGD 241 CLRYLTNTIE KFNGGVPGVY PVDLFEHIWA VDRLQRLGIS 281 RYFQTEIEEC MSYVYRYWTD KGICWARNSK VEDIDDTAMG 321 FRLLRLHGYM VSADVFAQFE KGGEFVCFAG QSNQALTGMF 361 NLYRASQVMF PGEKILADAK KFSSNFLHEK RANNELLDKW 401 IITKDLPGEV TYALDVPWYA SLPRVETRLY LEQYGGEDDV 441 WIAKTLYRMR KVNNKIYLEL GILDYNNCQA LHQLEWRSIQ 481 KWYKDSGLEE YGLSERNLLL AYYLATACIF EPERLVERLS 521 WAKTTALIYT TKSYFRTECN SGEQRKAFLH EFQQYCNDLD 561 YVSGARHKPT IRLIEALLGT LEQVSLDAIL DHGRYIHQDL 601 RNAWEKWLIA LQEGVDMDQE EAELTVLTLH LCAGSYTSEE 641 LLLSHPKYQQ LLNITSRVCH QIRQFQREKA QDTDNGRENL 681 VAITSIKAIE SDMQELAKLV LTKSTGDLAA KIKQTFLIVA 721 KSFYYTAHCL PGIISTHIAK VLFEKVF

A nucleic acid encoding the Chiococca alba (5R,8S,9S,10S)-labda-13-en-8-ol diphosphate (ent-8-LPP) synthase (CaTPS2) with SEQ ID NO:43 is shown below as SEQ ID NO:44.

1 ATGCCAGTAA TAAAGTCGCA TGAGTTTATT GAAGAGGTCG 41 GCCCGGAAAA AGGAACTCTG AAGCTGAGCA GATCAAGTAG 81 GATAAACGAA CTTGTAGAAT CAATTCAAAC GATGCTTCAA 121 TCGATGGATG ATGGGGAAAT AAGCATGTCT GCTTATGACA 161 CCGCGTGGGT TGCCCTTGTG GAAGATATTA ATGGAAGCAG 201 CTACCCTCAA TTCCCTATGA GCCTCGAGTG GATTGCCAAC 241 AATCAGCTTC CTGATGGTTC ATGGGGTGAC GGCAGTATCT 281 TTTCGGTTCA TGATCGGATA ATCAGCACAT TAGGATGTGT 321 TCTTGCATTA AAATCATGGA ACATGCACCC GGACAAAAGC 361 GAAAAAGGAC TGTTATTTAT AAGGGACAAT ATTCACAAGG 401 TTGGAGATGA CAGCGCTGAG CACATGCCTA TTGGTTTTGA 441 GGTGGTATTT CCTTCGCTTA TTGAGAGAGC CAAAAACTTG 481 GACATTGATA TTCCAGATAT TTCTGCTATC TTGCAAGAGA 521 TTTATGCACG AAGAAATCTA AAGCTCGCAA GGATTCCAAA 561 GGATATACTG TATACCGTGC CCACGACATT ACTTCATAGC 601 TTAGAAGGAA TGCCAGAACT GGACTGGCAA AAGCTACTGC 641 CATTAAAATG TGAGGATGGT TCATTTCTAT TTTCTCCATC 681 GTGCACTGCT TTTGCCCTCA TGCAGACTAA GGATGGTGAT 721 TGCCTCAGAT ATCTAACTAA TACCATAGAA AAATTCAATG 761 GGGGAGTTCC CGGTGTATAC CCTGTGGACT TGTTCGAACA 801 CATTTGGGCT GTTGATCGCT TGCAAAGACT AGGAATTTCC 841 CGGTATTTTC AGACAGAAAT TGAAGAATGT ATGAGTTATG 881 TTTACCGATA TTGGACGGAT AAAGGTATCT GTTGGGCTAG 921 AAACTCCAAA GTTGAAGACA TCGATGACAC AGCCATGGGT 961 TTTAGACTTC TAAGGTTGCA TGGTTACATG GTTTCTGCAG 1001 ATGTGTTTGC ACAGTTTGAG AAAGGGGGTG AATTCGTTTG 1041 CTTTGCTGGA CAGTCGAACC AGGCGCTGAC TGGAATGTTT 1081 AACCTGTATA GAGCTTCTCA AGTAATGTTT CCAGGGGAGA 1121 AGATACTTGC TGATGCCAAG AAATTCTCAT CGAACTTCTT 1161 ACATGAAAAG CGTGCAAACA ACGAGCTTCT AGATAAATGG 1201 ATCATAACTA AAGATTTGCC TGGAGAGGTG ACGTATGCGC 1241 TAGATGTTCC ATGGTACGCC AGTTTACCTC GTGTAGAAAC 1281 GAGATTATAT CTGGAACAAT ATGGAGGAGA GGATGATGTC 1321 TGGATTGCCA AGACATTGTA CAGGATGAGA AAAGTTAACA 1361 ACAAAATTTA CCTTGAACTT GGCATATTAG ATTACAATAA 1401 CTGTCAAGCA TTGCATCAGC TGGAGTGGAG AAGCATCCAA 1441 AAATGGTATA AGGATTCTGG CCTTGAAGAG TACGGGTTGA 1481 GCGAGAGGAA CCTTCTCCTG GCATATTATC TGGCCACAGC 1521 TTGTATATTT GAACCCGAAA GGTTGGTGGA GCGCCTTTCC 1561 TGGGCGAAAA CAACCGCCTT AATCTACACA ACAAAATCTT 1601 ATTTCAGAAC TGAATGCAAC TCTGGGGAAC AGAGAAAAGC 1641 TTTTCTTCAT GAGTTCCAAC AGTACTGCAA TGACCTGGAC 1681 TACGTTAGTG GCGCAAGGCA CAAGCCAACA ATAAGATTGA 1721 TCGAAGCTCT ACTTGGAACC CTAGAGCAGG TCTCTTTGGA 1761 TGCAATATTA GATCATGGCC GATATATCCA TCAAGATTTG 1801 CGTAATGCTT GGGAGAAATG GTTGATAGCT TTGCAAGAGG 1841 GAGTTGACAT GGACCAAGAA GAAGCAGAAC TTACAGTGCT 1881 CACACTACAC CTGTGTGCCG GCAGCTACAC ATCGGAGGAG 1921 TTACTGTTAT CTCATCCCAA GTATCAACAA CTTTTAAATA 1961 TCACTAGTAG AGTCTGCCAC CAAATTCGTC AATTCCAGCG 2001 CGAAAAGGCA CAGGATACGG ATAATGGAAG AGAAAACTTG 2041 CTTGCCATCA CAAGCATCAA GGCGATAGAA TCAGACATGC 2081 AAGAACTTGC GAAATTAGTT CTGACCAAAT CCACTGGCGA 2121 TTTAGCTGCT AAAATCAAGC AAACATTTCT TATAGTGGCA 2161 AAGAGCTTCT ACTACACCGC ACATTGCCTT CCTGGAATTA 2201 TCAGTACCCA CATTGCCAAA GTACTATTTG AGAAAGTTTT 2241 CTGA

A Chiococca alba CaTPS3 and CaTPS4 were identified and isolated. CaTPS3 and CaTPS4 were identified as an ent-kaurene synthase, converting ent-CPP [16] into ent-kaurene [19].

The Chiococca alba ent-kaurene synthase (CaTPS3) has the amino acid sequence shown below (SEQ ID NO:45).

1 MMMMMVVMNT APAHSYHPFP FAGPKSSATL FSNYYCSSRK 41 KSSPPRISAS VSLLTGVEST TAINSSDPEI KERIRKLFHD 81 VDISLSSYDT AWVAMVPAPH SSQSPLFPQC INWLLDNQLP 121 DGSWSLPPPH HHPLLLKDAL SSTLACVLAL RRWGIGQEQV 161 DKGIRFVELN FASASDQNQH LPVGFDIIFP GMLEYARDLN 201 LNLQLESATV NALLLKRDQE LTRFFKSYSD ESKAYLAYVS 241 EGIVKLQNWD TVMKFQRKNG SLFNSPSATA AAVMHVHNPG 281 CLDYLHSVLE KHGNAVPTVY PLDIYPRLCL VDNLERLGIC 321 GHFRKEILSV LDDTYRCWMQ GDEEIFAEKS TCAIAFTLLR 361 KHGYNISADP LTPFLKEECF SNSLGGCLKD TSAVLELYRA 401 LEMIISQNES ALVKKSLWSR SFLKEHISGG CDLKGFSNQI 441 SILVDDILNF PSHATLQRVA NRRSIEQYNL DSTKILKTSY 481 CSSNFSNKDL LILAVKDFNH CQLIHREELK ELERWVTDNR 521 LDKLKFARQK SAYCYFSAAA TIFSPELSDA RMSWAKNGVL 561 ATLVDDFFDV GGSLEELKKL IELVEKWDIN VSDGCCSEPV 601 QILFSALHST IQEIGDkAFK WQARSVTNHI FKIWLDLLNS 641 MLREAEWARN ATVPTVEEYM TNGYVSFALG PIILPALYLV 681 GPKLSEEVVK DSEFHSLFKL VSTCGRLLND VHSFERESKS 721 GQLNALSLRL IHGGVGITEA AAVAEMKSSI ENLRRELLRL 761 VLRKEGSVVP RACKDLFWNM SKVLHQFYNK DDGFTSEEMI 801 QLVKSIIYEP IAVNEFLNSC HT A nucleic acid encoding the Chiococca alba ent-kaurene synthase (CaTPS3) with SEQ ID NO:45 is shown below as SEQ ID NO:46.

1 ATGATGATCA TGATCGTGGT GATGAACACA GCTCCCGCCC 41 ACTCTTACCA TCCTTTCCCC TTTGCCGGCC CAAANTCCTC 81 AGCCACACTT TTTTCCAATT ATTATTGTTC CAGTAGGAAG 121 AAATCATCGC CACCTCGCAT CTCTGCCTCA GTTTCTTTGC 241 TAACTGGAGT TGAAAGCACA ACTGCAATTA ATTCTTCAGA 281 CCCGGAGATC AAAGAAAGAA TAAGGAAACT ATTTCATGAT 321 GTTGATATCT CGCTTTCTTC ATATGACACT GCATGGGTGG 361 CAATGGTCCC TGCTCCACAT TCTTCCCAGT CTCCCCTTTT 401 TCCCCAGTGC ATTAATTGGT TATTGGACAA TCAGCTTCCT 441 GATGGCTCAT GGAGTCTTCC TCCTCCTCAT CATCATCCTC 481 TATTACTTAA AGATGCATTA TCCTCTACCC TTGCATGTGT 521 TCTTGCGCTC AGGAGATGGG GAATTGGTCA AGAACAAGTT 561 GACAAGGGTA TTCGTTTTGT TGAGTTAAAT TTTGCTTCAG 601 CATCTGACCA GAACCAGCAT TTGCCACTTG GATTTGACAT 641 TATATTCCCT GGCATGCTCG AATATGCTAG AGATTTAAAT 681 TTAAATCTTC AACTAGAATC TGCAACAGTA AATGCCTTAC 721 TTCTTAAAAG AGATCAGGAG CTTACAAGAT TCTTTAAAAG 761 CTACTCAGAC GAGAGTAAAG CATACCTTGC ATATGTATCA 801 GAAGGTATAG TAAAGTTACA GAACTGGGAT ACAGTTATGA 841 AGTTCCAAAG AAAGAACGGG TCACTATTCA ATTCACCTTC 881 AGCTACAGCA GCTGCTGTTA TGCATGTCCA CAATCCTGGT 921 TGCCTCGATT ACCTTCACTC AGTGTTGGAG AAGCATGGAA 961 ATGCTGTTCC AACAGTTTAC CCTTTGGATA TATATCCACG 1001 CCTCTGCTTG GTTGACAACC TTGAGAGACT GGGTATTTGT 1041 GGTCATTTTA GGAAGGAAAT TCTGAGTGTA TTGGATGATA 1081 CATACAGATG CTGGATGCAG GGGGATGAAG AGATATTTGC 1121 AGAAAAATCA ACTTGTGCCA TAGCATTTAC ATTATTGCGA 1161 AAGCATGGGT ACAACATCTC TGCAGATCCA TTGACCCCAT 1201 TCTTAAAGGA AGAGTGTTTT TCCAATTCTT TGGGTGGATG 1241 TTTGAAAGAT ACTAGTGCTG TACTTGAATT ATACCGGGCA 1281 TTAGAGATGA TTATTAGCCA GAATGAATCA GCTCTGGTGA 1321 AAAAAAGCTT GTGGTCCAGA AGCTTCCTGA AAGAGCATAT 1361 TTCTGGTGGT TGTGATTTAA AGGGATTCAG CAATCAAATT 1401 TCCATACTGG TGGATGATAT CCTCAACTTT CCATCGCATG 1481 CTACTTTGCA ACGGGTTGCT AACAGGAGAA GCATAGAGCA 1521 ATACAACTTA GACAGTACAA AAATTTTAAA AACTTCATAT 1561 TGCTCGTCGA ATTTTAGCAA CAAAGATTTA TTGATCCTGG 1601 CAGTCAAAGA TTTTAATCAT TGCCAACTCA TACACCGTGA 1641 AGAACTGAAA GAACTAGAAA GGTGGGTCAC AGACAATAGA 1681 TTGGACAAGT TAAAGTTTGC TAGGCAGAAG TCTGCATACT 1721 GTTACTTTTC TGCTGCAGCA ACCATATTCT CACCTGAACT 1761 TTCTGATGCC CGCATGTCAT GGGCCAAGAA TGGTGTACTT 1801 GCTACTTTGG TTGATGACTT CTTTGACGTG GGAGGTTCTC 1841 TAGAGGAATT AAAGAAACTG ATTGACTTGG TTGAAAAGTG 1881 GGATATAAAT GTCAGTGATG GTTGTTGCTC TGAACCAGTG 1921 CAAATCCTCT TCTCAGCACT ACATAGTACA ATCCAGGAGA 1961 TTGGAGATAA AGCATTCAAA TGGCAAGCAC GCAGTGTAAC 2001 AAACCACATA TTTAAGATAT GGTTAGATTT GCTTAATTCT 2041 ATGTTGAGGG AAGCTGAGTG GGCTAGAAAT GCAACAGTGC 2081 CTACAGTTGA AGAATATATG ACAAATGGTT ATGTATCATT 2121 THCTTTGGGG CCAATTATCC TCCCTGCTCT TTATCTTGTT 2161 GGACCTAAGC TGTCAGAGGA AGTAGTTAAG GATTCTGAAT 2201 TCCACTCCCT TTTTAAGCTA GTGAGTACCT GTGGGCGGCT 2241 TCTGAATGAT GTCCACAGCT TCGAGAGGGA ATCAAAGTCC 2281 GGCCAACTAA ATGCTCTGTC TCTGCGCCTG ATTCATGGTG 2321 GTGTTGGCAT TACTGAAGCA GCTGCTGTTG CAGAGATGAA 2361 GAGTTCAATT GAGAATCTAA GGAGAGAACT GCTGAGACTA 2401 GTCTTGCGCA AAGAGGGTAG TGTAGTTCCA AGAGCTTGCA 2441 AGGATTTGTT TTGGAATATG AGTAAAGTGC TACATCAATT 2481 TTACAACAAA GATGATGGAT TTACTTCAGA GGAGATGATT 2521 CAGCTTGTGA AGTCGATCAT TTATGAGCCA ATTGCGGTCA 2561 ATGAATTTTT GAATAGTTGC CATACATGA

The Chiococca alba ent-kaurene synthase (CaTPS4) has the amino acid sequence shown below (SEQ ID NO:47).

1 MMIMVMNTAP VHAYHALPIP TQKSSTTLFP NYNCSSRKKS 41 SPPRISAASV SLQTGVERTT AIHSSDLEIK ERIRKLFHDV 81 DISLSSYDTA WVAKVPAPHS SQSPLFPQCI NWLLDNQLPD 121 GSWSLPPHHH HHHPLLLKDA LSSTLACVLA LRRWGIGQEQ 161 VDKGIRFVEL NFASASDQNQ HLPVGFDIIF PGMLEYARDL 201 NLNLQLESAT VDALLLKRDQ ELIRFFKSYS DESKAYLAYV 241 SEGIIKLQNW DTVMKFQRKN GSLFNSPSAT AAAVMHVHNP 281 GCLDYLHSVL EKHGNAVPTV YPLDIYPRLC LVDNLERLGI 321 CGHFRKEILS VLDDTYRCWM QGDEEIFAEK STCAIAFTLL 361 RKHGYNISAD PLTPFLKEEC FSNSLGGCLK DTSAVLELYR 401 ALEMIISQNE SALVKKSLWS RSFLKEHISG GCDLKGFSNQ 441 ISKQVDDILN FPSHATLQRV ANRRSIEQYN LDSTKILKTS 481 YCSSNFSNKD LLILAVKDFN HCQLIHREEL KELERWVADN 521 RLDKLKFARQ KSAYCYFSAA ATIFSPELSD ARISWAKNGV 561 LTTLVDDFFD VGGSLEELKK LIELVEKWDI NVSDGCCSEP 601 VQILFSALHS TIQEIGDKAF KWQARSVINH IIKIWLDLLN 641 SMLREAEWAR NATVPTVEEY MINGYVSFAL GPIILPALYL 681 VGPKLSEELV KDSEFHSLFK LVSTCGRLLN DVHSFERESK 721 AGQLNALSLR LIHGGVGITE AAAVAEMKSS IEKQRRELLR 761 LVLRKEGSVV PRACKDLFWN MSRVLHQFYV KDDGFTSEEM 801 IELVKSIIYE PIAVNEF A nucleic acid encoding the Chiococca alba ent-kaurene synthase (CaTPS4) with SEQ ID NO:47 is shown below as SEQ ID NO:48.

1 ATGATGATAA TGGTGATGAA CACAGCTCCC GTCCACGCTT 41 ACCACGCTTT ACCCATTCCC ACCCAAAAAT CCTCAACCAC 81 ACTTTTTCCC AATTATAACT GTTCCAGTAG GAAGAAATCA 121 TCGCCACCTC GCATCTCTGC CGCCTCAGTT TCTTTGCAAA 161 CTGGAGTTGA AAGAACGACG GCAATTCATT CTTCAGACCT 201 AGAGATCAAA GAAAGAATAA GGAAACTATT TCATGATGTT 241 GATATCTCGC TTTCTTCATA TGACACTGCA TGGGTGGCAA 281 TGGTCCCTGC TCCACATTCT TCCCAGTCTC CCCTTTTTCC 321 CCAGTGCATT AATTGGTTAT TGGACAATCA GCTTCCTGAT 361 GGCTCATGGA GTCTTCCTCC TCATCATCAT CATCATCATC 401 CCCTATTACT TAAAGATGCA TTATCCTCTA CGCTTGCATG 441 TGTTCTTGCG CTCAGGAGAT GGGGAATTGG TCAAGAACAA 481 GTTGACAAGG GTATTCGTTT TGTTGAGTTA AATTTTGCTT 521 CTGCATCTGA CCAGAACCAG CATTTGCCAG TTGGATTTGA 561 CATTATATTC CCTGGCATGC TCGAATATGC TAGAGATTTA 601 AATTTAAATC TTCAACTAGA ATCCGCAACT GTAGATGCCT 641 TACTTCTCAA AAGAGATCAG GAGCTTATAA GATTCTTTAA 681 AAGCTACTCA GACGAGAGTA AAGCATACCT TGCATATGTA 721 TCAGAAGGTA TCATAAAGTT ACAGAACTGG GATACAGTTA 761 TGAAGTTCCA AAGAAAGAAC GGGTCACTGT TCAATTCACC 801 TTCAGCTACA GCAGCTGCTG TTATGCATGT CCACAATCCT 841 GGCTGCCTCG ATTACCTTCA CTCAGTGTTG GAGAAGCATG 881 GCAATGCTGT TCCAACAGTT TACCCTTTGG ATATATATCC 921 ACGCCTCTGC TTGGTTGACA ACCTTGAGAG ACTGGGTATT 961 TGTGGTCATT TTAGGAAGGA AATTCTGAGT GTATTGGATG 1001 ATACATACAG ATGCTGGATG CAGGGGGATG AAGAGATATT 1041 TGCAGAAAAA TCAACTTGTG CCATAGCATT TACATTATTG 1081 CGAAAGCATG GGTACAACAT CTCTGCAGAT CCATTGACCC 1121 CATTCTTAAA GGAAGAGTGT TTTTCCAATT CTTTGGGTGG 1161 ATGTTTGAAA GATACTAGTG CTGTACTTGA ATTATACCGG 1201 GCATTAGAGA TGATTATTAG CCAGAATGAA TCAGCTCTGG 1241 TGAAAAAAAG CTTGTGGTCC AGAAGCTTCC TGAAAGAGCA 1281 TATTTCTGGT GGTTGTGATT TAAAGGGATT CAGCAATCAA 1321 ATTTCCAAAC AGGTGGATGA TATCCTCAAC TTTCCATCGC 1361 ATGCTACTTT GCAACGGGTT GCTAACAGGA GAAGCATAGA 1401 GCAATACAAC TTAGACAGTA CAAAAATTTT AAAAACTTCA 1441 TATTGCTCGT CGAATTTTAG TAACAAAGAT TTATTGATCC 1481 TGGCAGTCAA AGATTTTAAT CATTGCCAAC TCATACACCG 1521 TGAAGAACTG AAAGAACTAG AAAGGTGGGT CGCAGACAAT 1561 AGATTGGACA AGTTAAAGTT TGCTAGGCAG AAGTCTGCAT 1601 ACTGTTACTT TTCTGCTGCA GCAACCATAT TCTCACCTGA 1641 ACTTTCTGAT GCCCGCATCT CATGGGCCAA AAATGGTGTA 1681 CTTACTACTT TGGTTGATGA CTTCTTTGAC GTGGGAGGTT 1721 CTCTAGAGGA ATTAAAGAAA CTGATTGAGT TGGTTGAAAA 1761 GTGGGATATA AATGTCAGTG ATGGTTGTTG CTCTGAACCA 1801 GTGCAAATCC TCTTCTCAGC ACTACATAGT ACAATCCAGG 1841 AGATTGGAGA TAAAGCATTC AAATGGCAAG CACGCAGTGT 1881 AACAAACCAC ATAATTAAGA TATGGTTAGA TTTGCTTAAT 1921 TCTATGTTGA GGGAAGCTGA GTGGGCTAGA AATGCAACAG 1961 TGCCTACAGT TGAAGAATAT ATGACAAATG GTTATGTATC 2001 ATTTGCCTTG GGGCCAATTA TCCTCCCTGC TCTTTATCTT 2041 GTTGGACCTA AGCTCTCAGA GGAATTAGTT AAGGATTCTG 2081 AATTCCACTC CCTTTTTAAG CTAGTGAGTA CCTGTGGGCG 2121 GCTTCTGAAT GATGTCCACA GCTTCGAGAG GGAATCAAAG 2161 GCCGGCCAAC TAAATGCTCT TTCTCTGCGC CTGATTCATG 2201 GTGGAGTTGG CATTACTGAA GCAGCTGCTG TTGCAGAGAT 2241 GAAGAGTTCA ATTGAGAAGC AAAGGAGAGA ACTGCTGAGA 2281 CTAGTCTTGC GCAAAGAGGG TAGTGTAGTT CCAAGAGCTT 2321 GCAAGGATTT GTTTTGGAAT ATGAGTAGGG TGCTACATCA 2361 ATTTTACCTC AAAGATGATG GATTTACTTC AGAGGAGATG 2401 ATTGAGCTTG TGAACTCGAT CATTTATGAG CCAATTGCCG 2441 TCAATGAATT TTGA

A Chiococca alba 13(R)-epi-dolabradiene synthase (CaTPS5) was identified and isolated. This CaTPS5 enzyme was identified as an 13(R)-epi-dolabradiene synthase, which converts ent-CPP [16] to 13(R)-epi-dolabradiene.

The Chiococca alba 13(R)-epi-dolabradiene synthase (CaTPS5) has the amino acid sequence shown below (SEQ ID NO:49).

1 MIHTLPHGGQ AHFISHKTQP YYSSRPRFSS AASLDTRVRR 41 TSPSNSSVLD FNETKERITK LFHNVDYSIS SYDTAWVAMV 81 PDPHSSQAPL FPECINWLLD NQFHDGSWSL PHHNSLLLKD 121 VLSSTLACVL ALKRWGIGGR QIDKGVRFIE MNFGSASDNC 161 QHTPIGFDII FPGMLENARD LDLNLRLEPR IVIDMQRKRD 201 MQLTRLHESD LKGDQAYLAY VSEGMQKLQN WDLAMKFQRK 241 NGSLFNSPSA TAAAVMHVQN PASLNYLHSV VDKFGHAVPA 281 VYPLDLYARL CLVDNLERLG ICRHFTNEIE IVMEDTYRCW 321 LQDDEDIFAE ISTCALAFRL LRKHGYVVSP DPLTKIIEEE 401 DVSNSSGNGY WNDIHAVMEV HRASEVVIHE NESDLKNQNT 441 ISKHLLRHHL FNGSDVKPFP NPIYKQVDYA LKFPTPLILQ 481 RVENKTLIQN YDVDSTRLLK TSYRSSNFCN EDLLRLAVKD 521 FNDCQLLHRK ELKELERWSA DNRLHELKFA RQKAIYCSFS 561 AAATIFIPEW YEARMSLAKN SVLATVVDDF FDVGGSMEEL 601 KKLIEFVEKW DIDITKESCS EPLKIIFSAL HSTISEIGEQ 641 AVKWQGRNVT SHIIEIWLDL LNSMLRESEW TTDVHMPTLD 681 EYMEAAYVSF AMGPIIIPAL YFVGPKLSDE IVRDPEIRSL 721 HKLVSICGRL LNDMQGFERE KKAGKPNAVS IRISQNGDGI 761 TESAAFEEVK MELEDARREL LRLVVQKDGS VVPRACKDAF 801 WSVSRMLHHF YFNNDGYTSE VEMVELVNSI IHEPLK

A nucleic acid encoding the Chiococca alba 13(R)-epi-dolabradiene synthase (CaTPS5) with SEQ ID NO:49 is shown below as SEQ ID NO:50.

1 ATGATTCATA CTCTCCCTCA TGGCGGCCAG GCTCACTTCA 41 TTTCCCACAA AACACACCCT TATTATTCCA GTAGACCTCG 81 CTTTTCTTCA GCAGCTTCTT TGGACACACG AGTCCGGAGA 121 ACATCGCCCT CTAATTCCTC TGTCCTAGAC TTCAAGGAGA 161 CCAAAGAAAG AATCACAAAA TTATTTCATA ATGTTGATTA 201 TTCAATTTCT TCATATGATA CAGCATGGGT TGCTATGGTC 241 CCGGACCCAC ATTCTTCTCA GGCTCCCCTT TTCCCAGAGT 281 GCATAAATTG GTTGCTAGAT AATCAATTTC ATGATGGCTC 321 CTGGAGTCTT CCTCATCACA ATTCTCTATT GCTTAAGGAT 361 GTTTTATCCT CTACGCTTGC GTGTGTTCTT GCTCTTAAGA 401 GATGGGGAAT AGGAGGAAGG CAGATTGACA AAGGTGTTCG 441 CTTTATTGAG ATGAATTTTG GCTCAGCATC TGACAATTGC 481 CAGCATACTC CAATAGGATT TGACATAATA TTTCCAGGAA 521 TGCTTGAAAA TGCCAGAGAT TTGGATCTAA ATCTTAGACT 561 ACAACCCAGA ATTGTAACTG ACATGCAACG TAAAAGAGAC 601 ATGCAGCTTA CAAGACTCCA TGAAAGCGAT CTAAAGGGGG 641 ACCAAGCATA CTTGGCATAT GTATCCGAAG GGATGCAAAA 681 GTIACAGAAT TGGGATTTGG CGATGAAGTT TCAAAGGAAG 721 AATGGATCGC TCTTCAACTC ACCATCAGCT ACAGCAGCCG 801 CTGTTATGCA TGTCCAAAAT CCTGCTTCCC TCAATTATCT 841 TCATTCAGTC GTCGACAAAT TCGGCCATGC AGTTCCGGCT 881 GTTTACCCTT TGGATCTCTA TGCGCGCCTT TGCTTGGTTG 921 ACAATCTTGA GAGGCTGGGT ATCTGTCGAC ATTTTACTAA 961 TGAAATTGAA ATTGTAATGG AGGACACGTA CAGGTGCTGG 1001 CTGCAGGATG ATGAAGATAT ATTTGCCGAA ATATCAACTT 1041 GTGCCTTAGC TTTTCGGTTA TTGAGAAAAC ATGGCTATGT 1081 TGTCTCCCCA GATCCACTGA CAAAAATCAT AGAAGAAGAA 1121 GATGTTTCCA ATTCTTCTGG TAATGGATAT TGGAATGATA 1161 TACATGCTGT AATGGAAGTG CATCGGGCAT CAGAGGTGGT 1201 TATACATGAA AATGAATCAG ATTTAAAGAA TCAAAATACC 1241 ATATCAAAAC ACCTTCTCAG ACACCATCTT TTCAATGGTT 1281 CTGATGTGAA GCCCTTTCCT AATCCAATAT ACAAGCAGGT 1321 GGACTATGCT CTCAAGTTTC CAACCCCCTT AATTCTACAA 1361 CGTGTTGAAA ACAAGACCCT CATACAGAAC TACGACGTAG 1401 ACAGTACAAG ACTTCTTAAA ACTTCATATC GATCATCAAA 1441 TTTCTGCAAT GAAGATTTAC TGAGGTTAGC AGTGAAAGAT 1481 TTTAATGACT GTCAACTCCT GCACCGGAAA GAACTAAAAG 1521 AACTAGAAAG ATGGTCCGCA GATAACAGAC TGCACGAACT 1601 AAAAITTGCT CGGCAGAAAG CTATATACTG CTCCTTTTCT 1641 GCTGCAGCAA CGATTTTCAT ACCTGAATGG TACGAAGCCC 1681 GCATGTCATT GGCCAAAAAT AGTGTACTTG CTACTGTGGT 1721 TGATGACTTC TTTGATGTGG GTGGTTCGAT GGAGGAATTA 1761 AAGAAGCTAA TTGAATTTGT TGAAAAGTGG GATATTGACA 1801 TCACCAAGGA ATCCTGCTCT GAGCCACTCA AAATCATATT 1841 TTCAGCACTG CACAGTACAA TCTCTGAGAT TGGAGAGCAA 1881 GCAGTTAAAT GGCAAGGACG CAATGTAACA AGCCACATAA 1921 TTGAGATCTG GTTGGATTTG CTCAATTCGA TGTTGAGGGA 1961 GTCTCAATGG ACTACAGATG TGCACATGCC AACATTGGAT 2001 GAATATATGG AAGCTGCTTA TGTATCATTC GCCATGGGGC 2041 CAATTATCAT CCCTGCTCTG TATTTTGTTG GGCCTAAGCT 2081 ATCTGATGAA ATTGTTCGGG ATCCTGAAAT ACGATCCCTC 2121 CATAAGCTTG TGAGCATTTG TGGGCGGCTT CTAAATGATA 2161 TGCAAGGGTT CGAGAGGGAA AAGAAGGCTG GTAAACCAAA 2201 TGCCGTGTCT ATACGCATTA GTCAAAATGG TGATGGCATT 2241 ACCGAATCAG CAGCTTTCGA AGAAGTGAAG ATGGAATTAG 2281 AGGATGCAAG GAGAGAATTG CTAAGATTAG TTGTGCAAAA 2321 AGATGGTAGT GTAGTTCCAA GAGCTTGCAA GGATGCGTTT 2361 TGGAGCGTAA GCAGAATGTT GCATCATTTC TACTTCAATA 2401 ATGATGGATA CACGTCAGAG GTGGAGATGG TTGAGCTCGT 2441 GAATTCAATT ATTCATGAAC CACTAAAATA A

A Salvia hispanica (−)-kolavenyl diphosphate synthase (ShTPS1) was identified and isolated. This ShTPS1 enzyme was identified as an (−)-kolavenyl diphosphate synthase, which converts GGPP to (−)-kolavenyl diphosphate [36].

The Salvia hispanica (−)-kolavenyl diphosphate synthase (ShTPS1) has, for example, an amino acid sequence shown below (SEQ ID NO:51).

1 MSIQANMSFA TSLHRSTTPG VGLPLKPCIS PSPSLSFSPN 41 FGTFNNTSLR LKPEAGSKSY EGIRRSHQLA ASTILEGQTP 81 ITPEVESEKT RLIERIRSML QDMDNDGQIS VSPYDTAWVA 121 LVEDIGGSGG PQFPTSLEWI SNHQYDDGSW GDRKFVLYDR 161 ILNTLACVVA LTNWKMHPNK CEKGLRFIHE NIKKLADEDE 201 ELMPVGFEIA LPSVIDLAKR LGIEIPENSA SIKRIYELRD 241 SKLKKIPMDL VHKRPTSLLF SLEGMEGLNW DKLMNFLAEG 281 SFLSSPSSTA YALQHTKNEL CLEYLLKAVK RFNGGVPNAY 321 PVDMFEHLWS VDRLQRLGIS RYFQAEIEEN MAYAYRYWTN 361 KGITWARNMV VQDSDDSAQG FRLLRLYGYD IPIDVFKHFE 401 QGGQFCSIPG QMTHAITGMY NLYRASELLF PGEHILSDAR 441 KYTGNFLHQR RITNTVVDKW IITKDLHGEV AYALDVPFYA 481 SLPRLEARFF IEQYGGDEDV WIGKTLYRMF KVNSDTYLEM 521 AKLDYKQCQS VHQLEWNSMQ RLYRDCNLGE FGLSERSLLL 561 AYYIAASTTF EPEKSSERLA WAITTILVEI IASQKLSDEQ 601 KREFVDEFVK GSIVNNQNGG RHKPGNRLVE VLINNITLMA 641 EGRGTYQQLS NAWKKWLKTW EEGGDLGEAE ARLLLHTIHL 681 SSGLDDSSFS HPKYQQLLEA TSKVCHQLRV FQSVKVYDDQ 721 ESTSQLVTRT TFQIEAGMQE LVKLVFTKTL EDLPSTTKQS 761 FFSVARSFYY TACIHADTID SHINKVLFEK IV

A nucleic acid encoding the Salvia hispanica (−)-kolavenyl diphosphate synthase (ShTPS1) with SEQ ID NO:51 is shown below as SEQ ID NO:52.

1 ATGAGTATTC AAGCAAACAT GTCATTTGCC ACCTCCCTCC 41 ACCGATCAAC CACCCCCGGA GTTGGCCTTC CGCTAAAACC 81 ATGTATCTCT CCCTCTCCCT CTCTTTCCTT TTCCCCAAAC 121 TTTGGCACTT TTAACAACAC AAGTTTGAGA CTCAAACCAG 161 AGGCTGGGAG CAAAAGTTAT GAGGGGATTC GAAGAAGTCA 201 TCAATTAGCA GCATCAACAA TTTTGGAGGG TCAAACTCCG 241 ATTACTCCGG AGGTTGAATC GGAGAAAACA CGCCTGATTG 281 AAAGGATTCG TTCGATGTTA CAAGACATGG ACAACGATGG 321 CCAGATAAGT GTGTCACCAT ACGACACAGC ATGGGTGGCG 361 CTCGTGGAAG ATATTGGTGG CAGCGGAGGG CCACAGTTTC 401 CAACGAGCCT AGAGTGGATT TCTAACCACC AGTACGACGA 441 TGGATCGTGG GGGGATCGCA AATTTGTTCT CTATGACCGG 481 ATACTCAATA CATTAGCATG TGTTGTCGCA CTCACGAATT 521 GGAAAATGCA TCCTAACAAA TGCGAAAAAG GGTTGAGGTT 561 TATTCATGAG AATATTAAGA AACTCGCGGA TGAAGATGAA 601 GAGCTCATGC CCGTAGGATT CGAAATCGCA CTGCCATCAG 641 TCATTGATTT AGCTAAAAGA CTGGGTATAG AAATCCCAGA 681 AAATTCTGCA AGCATAAAAA GAATTTATGA ATTGAGAGAT 721 TCAAAACTTA AAAAAATACC AATGGATTTA GTGCACAAAA 761 GGCCCACATC ACTACTCTTC AGCTTGGAAG GCATGGAAGG 301 CCTTAACTGG GACAAACTAA TGAATTTTCT AGCCGAGGGT 841 TCGTTTCTTT CATCGCCATC GTCCACTGCC TACGCTCTCC 881 AACACACCAA GAATGAGTTA TGCCTAGAGT ATTTACTCAA 921 GGCAGTCAAG AGATTCAATG GTGGAGTTCC AAATGCATAC 961 CCTGTCGACA TGTTTGAGCA TCTGTGGTCC GTGGATCGCT 1001 TACAGAGATT AGGAATTTCT CGGTATTTTC AAGCTGAAAT 1041 TGAAGAAAAC ATGGCCTATG CTTACAGATA CTGGACAAAT 1081 AAAGGAATCA CCTGGGCAAG AAATATGGTT GTCCAAGACA 1121 GTGACGACAG CGCACAGGGA TTCAGGCTCT TAAGGTTGTA 1161 CGGATACGAT ATTCCTATAG ATGTTTTCAA ACATTTCGAG 1201 CAAGGTGGAC AATTCTGCAG CATACCAGGA CAGATGACAC 1241 ACGCTATTAC AGGAATGTAC AACTTGTATA GAGCTTCTGA 1281 ACTTCTGTTC CCTGGAGAAC ACATACTTTC TGATGCTAGA 1321 AAATACACAG GTAACTTCTT GCATCAAAGA AGAATTACTA 1361 ACACGGTAGT AGACAAGTGG ATCATTACCA AAGACCTTCA 1401 CGGCGAGGTG GCTTATGCAT TGGATGTGCC ATTCTACGCC 1441 AGTCTGCCAC GACTGGAAGC ACGATTCTTC ATAGAACAAT 1481 ATGGGGGTGA TGAAGATGTT TGGATTGGGA AAACATTGTA 1521 CAGGATGTTT AAAGTAAACT CCGACACATA CCTTGAGATG 1561 GCAAAATTAG ATTACAAACA ATGCCAGTCT GTGCATCAGT 1601 TAGAGTGGAA TAGCATGCAA AGATTGTATA GAGATTGCAA 1641 TCTAGGAGAG TTTGGGTTGA GCGAAAGAAG CCTTCTCCTA 1681 GCTTACTACA TAGCAGCCTC AACTACATTT GAGCCGGAAA 1721 AATCAAGTGA AAGACTGGCT TGGGCTATAA CAACAATTTT 1761 AGTCGAAATA ATCGCATCCC AAAAACTCTC TGATGAGCAA 1801 AAGAGAGAGT TTGTTGATGA ATTTGTAAAA GGAAGCATCG 1841 TCAATAACCA AAATGGAGGA AGACATAAAC CGGGAAACAG 1881 ATTGGTTGAA GTTTTGATCA ACAATATAAC ACTGATGGCA 1921 GAAGGCAGAG GCACATATCA GCAGTTGTCT AATGCGTGGA 1961 AAAAATGGCT AAAGACATGG GAAGAGGGAG GTGACCTGGG 2001 GGAAGCACAA GCACGGCTTC TCCTGCACAC GATACATTTG 2041 AGCTCCGGAT TGGATGATTC ATCATTTTCC CATCCAAAAT 2081 ATCAGCAGCT CTTGGAGGCA ACCAGCAAAG TCTGCCACCA 2121 ACTTCGCGTA TTCCAGAGTG TAAAGGTGTA TGATGACCAA 2161 GAGTCTACAA GCCAACTGGT AACTAGGACA ACTTTCCAAA 2201 TAGAAGCAGG CATGCAAGAA CTAGTGAAAT TAGTTTTCAC 2241 AAAAACCTTG GAAGATTTGC CTTCTACTAC CAAGCAAAGC 2281 TTTTTTAGTG TTGCTAGAAG TTTCTATTAC ACTGCCTGTA 2321 TTCATGCAGA CACTATAGAC TCCCACATAA ACAAAGTATT 2361 GTTTGAAAAA ATTGTCTAG

A Teucrium canadense cleroda-4(18),13E-dienyl diphosphate synthase (TcTPS1) was identified and isolated as described herein. This TcTPS1 enzyme was identified as a cleroda-4(18), 13E-dienyl diphosphate synthase, which converts GGPP to cleroda-4(18),13E-dienyl diphosphate [38]. In addition, the combination of TcTPS1 and SsSS enzymes generated neo-cleroda-4(18),14-dien-13-ol [37]. These compounds are shown below.

The Teucrium canadense cleroda-4(18), 13E-dienyl diphosphate synthase (TcTPS1) amino acid sequence is shown below as SEQ ID NO:53.

1 MSFASQATSL LLSSHNATAL PPLSAARLPP LTAGAAPFGR 41 ISFTTTSLRQ YKLVSRAQSQ EVDEIEKVTQ VVLEAEKDID 81 QEAKVRELVE NVRVKLQNIG EGGISISPYD TAWVALVEDV 121 GGSGRPQFPE SLDWISNHQF PDGSWGSHKF LYYDRVLCTL 161 ACIVALKTWN LHPHKFDKGL KFVRENIGKL ADEEDVHMPI 201 GFEVAFPSLI ETAKRKGIDI PEDFPGKKEI YAKRDLKLKK 241 IPMDILHKIP TPLLFSIEGI EGLDWQKLFK FRDHGSFLTS 281 PSSTAHALQQ TKDELCLKYL TNLVKKNNGG VPNAFPVDLF 321 DRNYTVDRLR RLGILRYFQP EIEECMKYVY REWDKRCISW 361 ARNTHVQDLD DTVQGFRNLR MHGYDVTLDV FKQFERCGEF 401 FSFHGQSSDA VLCMFNLYRA SQVLFPGEDM LADARKYAAN 441 YLHKRRVSNR VVDKWIINKD LPGEVAYGLD VPFYASLPRL 481 EARFYVEQYG GNDDVWIGKA LYRMLNVSCD TYLELAKLDY 521 NICQAVHQKE WKSFQKWHRD GEFGLDEKSL LLAYYIAAST 561 VFEPEKSLER LAWAKTAVLM EAILSQQLPS TKKHELVDEF 601 KHASILNNQN GGSYKTRTPL VETLVNAISE LSTTILLEQD 641 RDIHLQLSNA WLKWLSRWEA RGNLVEAEAE LLLQTLHLSN 681 GLEESSFSHP KYQQLLQVIS KVCHLLRLFQ KRKVHDPEGC 721 TTDIATGTTF QIEACMQQVV KLVFTKSSHD LDSVVKQRFL 761 DVARSFYYTA HCDPQVIQSH INKVLFEKVV

A nucleic acid encoding the Teucrium canadense Cleroda-4(18),13E-dienyl diphosphate synthase (TcTPS1) has with SEQ ID NO:53 is shown below as SEQ ID NO:54.

1 ATGTCATTTG CTTCCCAAGC CACCTCCCTC CTCCTTTCTT 41 CCCACAACGC CACCGCTCTT CCGCCTCTCT CTGCCGCCCG 81 CCTTCCGCCT CTCACTGCCG GTGCTGCTCC ATTCGGAAGA 121 ATATCATTTA CTACTACCTC TCTTCGGCAG TATAAACTGG 161 TGTCAAGAGC TCAAAGCCAA GAGGTGGATG AGATTGAAAA 201 AGTGACACAA GTGGTATTGG AGGCAGAAAA AGACATCGAT 241 CAAGAGGCGA AGGTAAGGGA GCTGGTGGAA AATGTCCGAG 281 TGAAGCTGCA AAATATCGGG GAAGGAGGGA TAAGCATATC 321 GCCGTACGAC ACCGCATGGG TGGCGCTGGT GGAGGATGTC 361 GGCGGCAGCG GCAGACCGCA GTTCCCGGAG AGCCTGGATT 401 GGATATCAAA CCACCAGTTC CCGGACGGGT CGTGGGGCAG 441 CCACAAATTC TTGTACTATG ACCGGGTTTT GTGCACGTTA 481 GCATGTATAG TTGCATTGAA AACTTGGAAT CTGCATCCTC 521 ACAAATTCGA CAAAGGGTTG AAATTCGTCA GAGAGAACAT 561 TGGAAAGCTC GCGGATGAAG AAGACGTGCA CATGCCGATT 601 GGGTTCGAAG TGGCATTCCC ATCACTTATA GAGACTGCAA 641 AGAGAAAAGG AATTGACATC CCGGAAGATT TCCCTGGCAA 681 GAAAGAAATC TATGCAAAAA GAGACCTAAA GCTGAAAAAG 721 ATACCTATGG ATATACTGCA CAAAATCCCC ACACCATTAC 761 TGTTCAGCAT AGAAGGGATA GAAGGCCTTG ATTGGCAGAA 801 GCTATTCAAA TTCCGCGATC ACGGCTCCTT CCTCACGTCC 841 CCGTCCTCAA CGGCCCACGC TCTCCAGCAA ACAAAGGACG 881 AGTTATGCCT CAAATATCTG ACCAATCTTG TCAAAAAGAA 921 CAATGGGGGA GTTCCAAATG CATTTCCGGT GGACCTATTT 961 GATCGTAACT ATACAGTAGA TCGCCTGAGG AGGCTGGGAA 1001 TTTTGCGCTA TTTTCAACCT GAAATCGAGG AATGCATGAA 1041 ATATGTATAC AGATICTGGG ATAAAAGAGG AATCAGCTGG 1081 GCAAGAAATA CCCATGTTCA GGACCTTGAT GATACCGTAC 1121 AGGGATTCAG GAACTTAAGG ATGCATGGTT ATGATGTCAC 1161 CTTAGATGTT TTCAAACAGT TCGAGAGATG TGGAGAATTC 1201 TTTAGCTTCC ACGGGCAATC AAGTGATGCT GTCTTAGGAA 1241 TGTTCAACTT GTACCGAGCT TCTCAGGTTC TGTTTCCAGG 1281 AGAAGACATG CTTGCAGATG CAAGGAAGTA CGCGGCCAAC 1321 TATTTGCATA AAAGAAGAGT TAGTAATAGG GTCGTGGACA 1401 AATGGATTAT TAACAAAGAT CTTCCAGGCG AGGTGGCGTA 1441 TGGGCTAGAT GTTCCGTTCT ACGCCAGTCT ACCTCGACTG 1481 GAAGCAAGAT TCTACGTCGA ACAATATGGG GGTAACGATG 1521 ATGTCTGGAT TGGAAAAGCT TTATATAGAA TGTTGAATGT 1601 GAGCTGTGAT ACTTACCTTG AGCTAGCAAA ATTAGACTAC 1641 AATATTTGCC AGGCTGTGCA TCAGAAAGAG TGGAAAAGCT 1681 TTCAAAAATG GCACAGGGAT GGGGAGTTTG GATTGGATGA 1721 AAAAAGCTTA CTTTTAGCTT ACTACATAGC AGCCTCGACT 1761 GTTTTCGAGC CTGAAAAATC TCTAGAGCGA CTGGCTTGGG 1801 CTAAAACCGC AGTTCTAATG GAGGCAATTT TGTCCCAACA 1841 ACTTCCTAGC ACAAAAAAAC ATGAGCTTGT TGACGAATTT 1881 AAACATGCAA GCATCCTCAA CAACCAAAAT GGAGGAAGCT 1921 ATAAAACAAG AACTCCTTTG GTAGAGACTC TAGTAAACGC 1961 CATAAGTGAG CTCTCAACTA CCATACTATT GGAGCAAGAC 2001 AGAGACATTC ATCTGCAATT ATCTAATGCG TGGCTGAAGT 2041 GGCTAAGTAG ATGGGAGGCA AGAGGCAACC TAGTGGAAGC 2081 AGAAGCAGAG CTTCTTCTGC AAACCTTACA TCTGAGCAAT 2121 GGATTAGAAG AATCATCATT TTCTCATCCA AAATATCAAC 2161 AACTCTTACA GGTTACCAGC AAAGTCTGTC ACCTACTTCG 2201 GCTATTCCAG AAACGAAAGG TGCATGATCC GGAAGGGTGT 2241 ACAACAGACA TTGCAACAGG GACAACTTTC CAAATAGAAG 2281 CATGCATGCA ACAAGTAGTG AAATTAGTGT TCACCAAATC 2321 CTCACATGAT TTAGATTCTG TTGTTAAGCA GAGATTTTTG 2361 GATGTTGCCA GAAGTTTCTA TTACACAGCC CACTGTGATC 2401 CACAAGTGAT CCAGTCCCAC ATTAATAAAG TGTTGTTTGA 2441 AAAAGTAGTC TAG

Salvia officinalis (SoTPS2), Scutellaria baicalensis SbTPS1, and SbTPS2 enzymes were identified and isolated. These SoTPS2, SbTPS1, SbTPS2, CfTPS18a and CfTPS18b enzymes were all identified as ent-CPP synthases, which convert GGPP to ent-CPP.

The Salvia officinalis (SoTPS2) enzyme can have the amino acid sequence shown below (SEQ ID NO:55).

1 MSFASTTSLL RPSVTGFGVS PRVTSTSILS RSYGQILKGK 41 TKYITDNRRN RQLAVKFEGQ IALDLEDGVA KQTNQEAESE 81 KIRQLKGKIR WILQNMEDGE MSVSPYDTAW VALVEDISGG 121 GGPQFPTSLE WISKNQLADG SWGDPNYFLL YDRILNTLAC 161 VVALTTWNMH PHKCDQGLRF IRDNIEKLED EDEELILVGF 201 EIALPSLIDY AQNLGIQIQY DSPFIKKICA KRDLKLRKIP 241 MDLMHRKPTS LLYSLEGMEG LEWEKLMNLR SEGSFLSSPS 281 STAYALQHTK DELCLDYLVK AVNKFNGGVP NVYPVDMYEH 321 LWCVDRLQRL GISRYFQLEI QQCLDYVYRY WTNEGISWAR 361 YTNIRDSDDT AMGFRLLRLY GYDVSIDAFK PFEESGEFYS 401 MAGQMNHAVT GMYNLYRASQ LMFPQEHILS DARNFSAKFL 441 HQKRRTNALV DKWIITKDLP GEVGYALDVP FYASLPRLEA 481 RFFLEQYGGD DDVWIGYTLY RMPYVNSNTY LELAKVDYKN 521 CQSVHQLEWK SMQKWYRECN IGEFGLSERS LLLAYYIAAS 561 TTFEPEKSGE RLAWATTAIL IETIASQQLS DEQKREFVDE 601 FENSIIIKNQ NGGRYKARNR LVKVLINTVT LVAEGRGINQ 641 QLFNAWQKWL KTWEEGGDMG EAEAQLLLRT LHLSSGFDQS 681 SFSHPKYEQL LEATSKVCHQ LRLFQNRKVD DGQGCISRLV 721 IGTTSQIEAG MQEVVKLVFT KTSQDLTSAT KQSFFNIARS 761 FYYTAYFHAD TIDSHIYKVL FQTIV A nucleic acid encoding the Salvia officinalis (SoTPS2) has with SEQ ID NO:55 is shown below as SEQ ID NO:56.

1 ATGTCATTTG CTTCCACCAC CTCCCTCCTC CGACCAAGCG 41 TCACTGGGTT CGGTGTTTCT CCAAGGGTTA CTTCCACCTC 81 CATTCTTAGC CGAAGTTATG GTCAAATATT AAAAGGAAAA 121 ACAAAATACA TAACTGATAA CCGTAGAAAT CGACAATTGG 161 CGGTAAAATT TGAGGGCCAA ATTGCTTTGG ATTTGGAGGA 201 TGGCGTAGCA AAGCAGACGA ATCAAGAGGC GGAATCTGAG 241 AAGATAAGGC AACTGAAGGG AAAGATCCGA TGGATTCTGC 281 AAAACATGGA GGACGGCGAG ATGAGCGTGT CGCCGTACGA 321 CACCGCATGG GTGGCGCTGG TGGAAGATAT CAGCGGCGGC 361 GGCGGGCCGC AGTTCCCGAC GAGCCTCGAG TGGATTTCCA 401 AGAATCAGTT GGCGGATGGG TCATGGGGGG ATCCTAATTA 441 TTTCCTTCTC TACGACAGAA TACTCAATAC TTTAGCATGT 481 GTAGTCGCAC TCACGACTTG GAATATGCAT CCTCACAAAT 521 GCGATCAAGG GTTGAGGTTT ATAAGAGACA ACATTGAGAA 561 ACTTGAGGAT GAAGATGAGG AGCTAATTCT CGTAGGATTC 601 GAGATCGCAC TGCCTTCACT CATTGATTAT GCTCAAAACC 641 TTGGGATACA AATCCAATAT GATTCTCCAT TCATTAAAAA 681 AATTTGTGCA AAGAGAGATC TAAAACTCAG AAAAATACCA 721 ATGGATTTAA TGCACAGAAA GCCAACATCA TTGCTCTACA 761 GCTTGGAAGG CATGGAAGGC CTTGAGTGGG AAAAGCTAAT 801 GAATTTGCGA TCGGAGGGTT CGTTTCTGTC ATGGCCGTCG 841 TCCACGGCCT ACGCTCTCCA ACACACCAAG GATGAGTTAT 881 GCCTTGACTA TCTGGTCAAG GCGGTCAACA AATTCAATGG 921 TGGAGTTCCC AACGTGTACC CTGTCGACAT GTATGAGCAT 961 CTATGGTGCG TAGACCGCTT GCAGAGGTTG GGAATTTCTC 1001 GCTATTTTCA ACTTGAAATT CAACAATGCC TCGACTATGT 1041 TTACAGATAC TGGACAAATG AAGGAATTTC GTGGGCAAGA 1081 TATACTAATA TCCGGGATAG TGACGACACC GCAATGGGAT 1121 TCAGGCTTCT AAGGTTGTAC GGCTATGATG TCTCTATAGA 1161 TGCTITTAAA CCATTCGAGG AAAGCGGAGA ATTCTATAGC 1201 ATGGCAGGGC AGATGAACCA CGCTGTTACA GGAATGTACA 1241 ACTTGTACAG AGCTTCTCAA CTTATGTTCC CTCAAGAACA 1281 CATACTTTCC GATGCCAGAA ACTTCTCTGC CAAATTCTTG 1321 CATCAAAAGA GGCGTACTAA TGCACTAGTA GACAAGTGGA 1361 TCATTACCAA AGACCTTCCC GGCGAGGTTG GATATGCATT 1401 GGATGTGCCG TTCTACGCCA GTCTGCCTCG ACTGGAAGCA 1441 CGATTCTTCT TAGAACAATA TGGGGGTGAT GATGATGTTT 1481 GGATTGGAAA AACTTTGTAC AGGATGCCAT ATGTGAACTC 1521 CAACACATAC CTTGAGCTTG CAAAAGTAGA CTACAAAAAC 1561 TGCCAGTCCG TGCATCAGTT GGAGTGGAAG AGCATGCAAA 1601 AATGGTACAG AGAATGCAAT ATAGGTGAGT TTGGGTTGAG 1641 CGAAAGAAGC CTTCTCCTAG CTTACTACAT AGCAGCCTCA 1681 ACTACATTCG AGCCAGAAAA ATCAGGTGAG CGGCTCGCTT 1721 GGGCTACAAC AGCAATTTTA ATCGAGACAA TCGCGTCCCA 1761 ACAACTCTCC GATGAACAAA AGAGAGAGTT CGTTGATGAA 1801 TTTGAAAACA GCATCATTAT CAAGAATCAA AATGGAGGGA 1841 GATATAAAGC AAGAAACAGA TTGGTCAAGG TTTTGATCAA 1381 CACTGTAACA CTGGTAGCAG AAGGCAGAGG CATAAATCAG 1921 CAGTTGTTTA ATGCGTGGCA AAAATGGCTA AAGACATGGG 1961 AAGAAGGAGG TGACATGGGG GAAGCAGAAG CCCAGCTTCT 2001 TCTGCGCACG CTACATTTGA GCTCCGGATT CGATCAATCA 2041 TCATTTTCCC ATCCAAAATA TGAGCAGCTC TTGGAGGCGA 2081 CCAGCAAAGT TTGCCACCAA CTTCGCCTAT TCCAGAATCG 2121 AAAGGTGGAT GATGGCCAAG GGTGTATAAG TCGATTGGTA 2161 ATTGGGACAA CTTCCCAAAT AGAAGCAGGC ATGCAAGAAG 2201 TAGTGAAATT AGTTTTCACC AAAACCTCAC AAGACTTGAC 2241 TTCTGCTACC AAGCAAAGCT TTTTCAATAT TGCTAGAAGT 2281 TTCTATTATA CTGCCTACTT TCATGCAGAC ACTATAGACT 2321 CCCACATATA CAAAGTATTG TTTCAAACAA TAGTATAG

A Scutellaria baicalensis SbTPS1 amino acid sequence shown below (SEQ ID NO: 57).

1 MPFLLPSSAT SSPAFYTPAA PLAGHHVFPS FKPLIISRSS 41 LQCNAISRPR TQEYIDVIQN GLPVIKWHEA VEEDETDKDS 81 LNKEATSDKI RELVNLIRSM LQSMGDGEIS SSPYDAAWVA 121 LVPDVGGSGG PQFPSSLEWI SKNQLPDGSW GDTCTFSIYD 161 RIINTLACVV ALKSWNIHPH KTYQGISFIK ANMDKLEDEN 201 EEHMPIGFEV ALPSLIEIAK RLDIDISSDS RGLQEIYTRR 241 EVKLKRIPKE IMHQVPTTLL HSLEGMAELT WHKLLKLQCQ 281 DGSFLFSPSS TAFALHQTKD HNCLHYLTKY VHKFHGGVPN 321 VYPVDLFEHL WAVDRIQRLG ISRHFKPQVD ECIAYVYRYW 361 TDKGICWARN SVVQDLDDTA MGFRLLRLHG YDVSADVFKH 401 FENGGEFFCF KGQSTQAVTG MYNLYRASQL MFPGESILED 441 AKTESSKFLQ RKRANNELLD KWIITKDLPG EVGYALDVPW 481 YASLPRVETR FYLEQYGGED DVWIGKTLYR MPYVNNNKYL 521 ELAKLDYSNC QSLHQQEWKN IQKWYESCNL GEFGLSERRV 561 LLAYYVAAAC IYEPEKSNQR LAWAKTVILM ETITSYFEHQ 601 QLSAEQRRAF VNEFEHGSIL KYANGGRYKR RSVLGTLLKT 641 LNQLSLDILL THGRNVHQPF KNAWHKWLKT WEEGGDIEEG 681 EAEVLVRTLN LSGEGRHDSY VLEQSLLSQP IYEQLLKATM 721 SVCKKLRLFQ HRKDENGCMT KMRGITTLEI ESEMQELVKL 761 VFTKSSDDLD CEIKQNFFTI ARSFYYVAYC NQGTINYHIA 801 KVLFERVL A nucleic acid encoding the Scutellaria baicalensis SbTPS1 with SEQ ID NO:57 is shown below as SEQ ID NO:58.

1 ATGCCTTTCC TCCTCCCTTC CTCCGCCACC AGCTCCCCCG 41 CGTTCTATAC TCCGGCCGCG CCTCTCGCCG GTCATCATGT 31 TTTTCCATCT TTCAAGCCAC TCATTATTTC CCGTTCTTCA 121 CTCCAATGCA ATGCAATCTC TCGACCTCGT ACCCAAGAAT 161 ACATAGATGT GATTCAGAAT GGATTGCCAG TAATAAAGTG 201 GCACGAAGCT GTGGAAGAAG ATGAGACAGA TAAAGATTCT 241 CTTAATAAGG AGGCCACGTC AGACAAGATA AGAGAGTTGG 281 TAAATCTGAT CCGTTCGATG CTCCAATCAA TGGGCGACGC 521 AGAGATAAGC TCGTCGCCGT ACGACGCCGC ATGGGTGGCG 561 CTGGTGCCGG ACGTCGGCGG CTCCGGCGGG CCCCAGTTCC 601 CCTCCAGCCT CGAATGGATA TCCAAAAACC AACTCCCCGA 641 CGGCTCCTGG GGCGACACGT GTACCTTTTC CATTTATGAT 681 CGAATCATCA ACACACTGGC TTGCGTTGTT GCTTTGAAAT 721 CTTGGAACAT ACATCCCCAC AAAACTTATC AAGGGATTTC 761 ATTCATAAAG GCAAATATGG ACAAACTTGA AGACGAGAAC 801 GAGGAGCACA TGCCGATCGG ATTTGAAGTG GCACTCCCGT 841 CGCTAATCGA GATAGCGAAA AGGCTCGATA TCGATATTTC 881 CAGCGATTCG AGAGGGCTGC AAGAGATATA CACGAGGAGG 921 GAGGTAAAGC TGAAAAGGAT ACCGAAAGAG ATAATGCACC 961 AAGTGCCCAC AACACTGCTT CATAGCTTGG AGGGTATGGC 1041 CGAGCTGACG TGGCACAAGC TTTTGAAATT ACAGTGCCAA 1081 GATGGCTCCT TTCTTTTCTC TCCATCTTCA ACTGCCTTTG 1121 CTCTTCACCA AACTAAGGAC CATAATTGTC TCCATTATTT 1161 GACCAAATAT GTTCACAAAT TTCATGGTGG AGTGCCAAAT 1201 GTGTATCCGG TGGACTTGTT CGAGCATCTA TGGGCAGTTG 1241 ATCGGATCCA ACGGCTGGGG ATTTCCCGGC ATTTCAAGCC 1281 CCAAGTTGAT GAATGTATTG CCTATGTTTA TAGATATTGG 1321 ACAGATAAAG GAATATGCTG GGCAAGAAAT TCAGTAGTTC 1361 AAGATCTTGA TGACACAGCC ATGGGATTCA GGCTTCTTAG 1401 GTTGCATGGC TACGATGTTT CAGCAGATGT TTTCAAACAT 1441 TTTGAAAATG GTGGAGAGTT CTTCTGCTTC AAAGGGCAAA 1481 GCACGCAGGC AGTGACTGGA ATGTACAATC TGTACAGAGC 1521 TTCTCAGTTG ATGTTTCCTG GAGAAAGCAT ACTGGAAGAT 1601 GCTAAGACCT TCTCATCTAA GTTTTTGCAA CGAAAACGAG 1641 CCAATAACGA GTTGTTAGAT AAGTGGATTA TTACCAAGGA 1681 TCTTCCTGGA GAGGTGGGAT ATGCTCTAGA TGTACCATGG 1721 TATGCTAGCT TACCTAGAGT TGAAACTAGA TTCTACTTGG 1801 AACAATATGG TGGTGAAGAT GATGTTTGGA TTGGCAAAAC 1841 TTTATACAGG ATGCCATATG TTAACAATAA TAAATATCTA 1881 GAACTGGCAA AATTAGACTA TAGTAACTGC CAGTCATTAC 1921 ATCAACAAGA GTGGAAAAAC ATTCAAAAAT GGTATGAGAG 1961 TTGCAATCTG GGAGAATTTG GTITGAGTGA AAGAAGGGTT 2001 CTACTAGCCT ACTACGTAGC TGCTGCCTGT ATATATGAGC 2041 CCGAAAAGTC AAACCAGCGC TTGGCTTGGG CCAAAACCGT 2081 AATTTTAATG GAGACTATTA CTTCCTATTT TGAGCACCAA 2121 CAACTCTCCG CAGAACAGAG ACGCGCCTTT GTTAATGAAT 2161 TTGAACATGG GAGTATCCTC AAATATGCAA ATGGAGGAAG 2201 ATACAAAAGG AGGAGTGTTT TGGGGACTTT GCTCAAAACA 2241 CTAAATCAGC TTTCATTGGA TATATTATTG ACACACGGTC 2281 GAAACGTCCA TCAGCCTTTC AAAAATGCGT GGCACAAGTG 2321 GCTAAAAACG TGGGAAGAAG GAGGTGACAT TGAAGAAGGC 2361 GAAGCAGAGG TATTGGTCCG AACCCTAAAC CTAAGCGGCG 2401 AAGGGAGGCA CGACTCCTAT GTATTGGAGC AATCATTATT 2441 GTCAGAACCT ATATATGAAC AACTTTTGAA AGCCACCATG 2481 AGTGTTTGCA AGAAGCTTCG ATTGTTCCAA CATCGAAAGG 2521 ATGAGAATGG ATGTATGACG AAGATGAGAG GCATTACAAC 2561 GTTAGAGATA GAATCGGAGA TGCAAGAATT AGTGAAATTA 2601 GTATTTACTA AATCCTCAGA TGATTTAGAT TGTGAAATTA 2641 AACAAAACTT TTTTACAATT CGTAGGAGTT TCTATTATGT 2681 GGCTTATTGT AACCAAGGAA CTATCAACTT TCACATTGCT 2721 AAGGTGCTCT TTGAAAGAGT TCTTTAG

A Scutellaria baicalensis SbTPS2 amino acid sequence is shown below (SEQ ID NO:59).

1 MASLSTLSLN FSPAIHRKIQ QSSAKLQFQG HCFTISSCMN 41 NSKRLSLNHQ SNHKRTSNVS ELQVATLDAP QIREKEDYST 81 AQGYEKVDEV EDPIEYIRML LNTTGDGRIS VSPYDTAWIA 121 LIKDVEGRDA PQFPSSLEWI ANNQLSDGSW GDEKFFCVYD 161 RLVNTLACVV ALRSWNIDAE KSEKGIRYIK ENVDKLKDGN 201 PEHMTCGFEV VEPSLLQRAQ SMGIHDLPYD APVIQDIYNT 241 RESKLKRIPM EVMHKVPTSL LFSLEGLENL EWDKLLKLQS 281 SDGSFLTSPS STAYAFMHTK DPKCFEFIKN TVETFNGGAP 321 HTYPVDVFGR LWAIDRLQRL GISRFFESEI ADCLDHIYKY 361 WTDKGVFSGR ESDFVDVDDT SMGVRLLRMH GYQVDPNVLR 401 NFKQGDKFSC YGGQMIESSS PIYNLYRASQ LRFPGEDILE 441 DANKFAYEFL QEQLSNNQLL DKWVISKHLP DEIKLGLQMP 481 WYATLPRVEA KYYLQYYAGA DDVWIGKTLY RMPEISNDTY 521 LELARMDFKR CQAQHQFEWI SMQEWYESCN IEEFGISRKE 561 LLQAYFLACS SVFELERTTE RIGWAKSQII SRMIASFFNN 601 ETTTADEKDA LLTRFRNING PNRTKSGQRE SEAVNMLVAT 641 LQQYLAGFDR YTRHQLKDAW SVWFRKVQEE EAIYGAEAEL 681 LTTTLNICAG HIAFDENIMA NYDYTTLSSL TSKICQKLSE 721 IRNEKVEEME SGIKAKSSIK DKEVEHDMQS LVKLVLERCE 761 GINNRKLKQT FLSVAKTYYY RAYNADETMD IHMFKVLFEP 801 VM A nucleic acid encoding the Scutellaria baicalensis SbTPS2 with SEQ ID NO:59 is shown below as SEQ ID NO:60.

1 ATGGCCTCTC TATCAACTCT GAGCCTCAAC TTTTCCCCAG 41 CAATTCACCG CAAAATACAG CAATCATCTG CAAAACTTCA 81 GTTCCAGGGA CATTGTTTCA CCATAAGTTC ATGCATGAAC 121 AACAGTAAAA GACTGTCTTT GAACCACCAA TCTAATCACA 161 AAAGAACGTC AAACGTATCT GAGCTGCAAG TTGCCACTTT 201 GGATGCGCCC CAAATACGTG AAAAAGAAGA CTACTCCACT 241 GCTCAAGGCT ATGAGAAGGT GGATGAAGTA GAGGATCCTA 281 TCGAATATAT TAGAATGCTG TTGAACACAA CAGGTGATGG 321 GCGAATAAGT GTGTCGCCAT ACGACACAGC CTGGATCGCT 361 CTTATTAAAG ACGTGGAAGG ACGTGATGCT CCCCAGTTCC 401 CATCTAGTCT CGAATGGATT GCCAATAATC AACTGAGTGA 441 TGGGTCGTGG GGCGATGAGA AGTTTTTCTG TGTGTATGAT 481 CGCCTTGTTA ATACACTTGC ATGTGTCGTG GCATTGAGAT 521 CATGGAATAT TGATGCTGAA AAGAGCGAGA AAGGAATAAG 561 ATACATAAAA GAAAACGTGG ATAAACTGAA AGATGGGAAT 601 CCAGAGCACA TGACCTGTGG TTTTGAGGTG GTGTTTCCTT 641 CCCTTCTTCA GAGAGCCCAA AGTATGGGAA TTCATGATCT 681 TCCCTATGAT GCTCCTGTCA TCCAAGACAT TTACAATACC 721 AGGGAGAGTA AATTGAAAAG CATTCCAATG GAGGTTATCC 761 ACAAGGTGCC AACATCTCTA TTGTTCAGCT TGGAAGGATT 801 GGAGAATTTG GAGTGGGATA AGCTCCTCAA ACTTCAGTCT 841 TCTGATGGTT CATTCCTCAC TTCTCCATCC TCAACTGCCT 881 ATGCTTTCAT GCACACTAAG GACCCTAAAT GCTTCGAATT 921 CATCAAAAAC ACCGTCGAAA CATTTAATGG AGGAGCACCT 961 CATACTTATC CGGTGGATGT TTTTGGAAGA CTGTGGGCCA 1001 TTGACAGGCT GCAGCGCCTC GGAATCTCTC GCTTCTTTGA 1041 GTCCGAGATT GCTGATTGCT TAGATCACAT CTATAAATAT 1081 TGGACAGACA AAGGAGTGTT CAGTGGAAGA GAATCAGATT 1121 TTGTGGATGT GGATGACACA TCCATGGGTG TTAGGCTTCT 1161 AAGGATGCAC GGATATCAAG TTGATCCAAA TGTATTGAGG 1201 AACTTCAAGC AGGGTGACAA ATTTTCATGC TATGGTGGTC 1241 AAATGATAGA GTCATCATCT CCGATATACA ATCTCTATAG 1281 GGCTTCTCAA CTCCGATTTC CAGGAGAAGA CATTCTTCAA 1321 GATGCCAACA AATTCGCATA CGAGTTCTTG CAAGAACAGC 1361 TATCCAACAA TCAACTTTTG GACAAATGGG TTATATCCAA 1401 GCACTTGCCT GATGAGATAA AGCTTGGATT GCAGATGCCA 1441 TGGTATGCCA CCCTACCCCG AGTGGAGGCT AAATACTACC 1481 TACAGTATTA TGCTGGTGCT GATGATGTCT GGATCGGCAA 1521 GACTCTCTAC AGAATGCCAG AAATCAGTAA TGATACATAT 1561 CTGGAGTTAG CAAGAATGGA TTTCAAGAGA TGCCAAGCAC 1601 AGCATCAATT TGAGTGGATT TCCATGCAAG AATGGTATGA 1641 AAGTTGCAAC ATTGAAGAAT TTGGGATAAG CAGLAAAGAG 1681 CTTCTTCAGG CTTACTTTTT GGCCTGCTCA AGTGTATTTG 1721 AACTCGAGAG GACAACAGAG AGAATAGGAT GGGCCAAATC 1761 CCAAATTATT TCAAGGATGA TAGCTTCTTT CTTCAACAAT 1801 GAAACTACAA CAGCCGATGA AAAAGATGCA CTTTTAACCA 1841 GATTCAGAAA CATCAATGGC CCAAACAAAA CAAAAAGTGG 1881 TCAGAGAGAG AGTGAAGCTG TGAACATGTT GGTAGCAACG 1921 CTCCAACAAT ACCTGGCAGG ATTTGATAGA TATACCAGAC 1961 ATCAATTGAA AGATGCTTGG AGTGTGTGGT TCAGAAAAGT 2001 GCAAGAAGAA GAGGCCATCT ACGGGGCAGA AGCGGAGCTT 2041 CTAACAACCA CCTTAAACAT CTGTGCTGGT CATATTGCTT 2081 TCGACGAAAA CATAATGGCC AACAAAGATT ACACCACTCT 2121 TTCCAGCCTT ACAAGCAAAA TTTGCCAGAA GCTTTCTGAA 2161 ATTCGAAATG AAAAGGTTGA GGAAATGGAG AGTGGAATTA 2201 AAGCAAAATC AAGCATCAAA GACAAGGAAG TGGAACATGA 2241 TATGCAGTCA CTGGTGAAAT TAGTCCTGGA GAGATGTGAA 2281 GGCATAAACA ACAGAAAACT GAAGCAAACA TTTCTATCGG 2321 TTGCAAAAAC ATATTACTAC AGAGCCTATA ATGCTGATGA 2361 AACCATGGAC ATCCATATGT TCAAAGTACT TTTCGAACCA 2401 GTCATGTGA

An example of a Salvia sclarea sclareol synthase amino acid sequence is shown below (SEQ ID NO:176; NCBI accession no. AET21246.1).

1 MSLAFNVGVT PFSGQRVGSR KEKFPVQGFP VTTPNRSRLI 41 VNCSLTTIDF MAKMKENFKR EDDKFPTTTT LRSEDIPSNL 81 CIIDTLQRLG VDQFFQYEIN TILDNTFRLW QEKHKVIYGN 121 VTTHAMAFRL LRVKGYEVSS EELAPYGNQE AVSQQTNDLP 161 MIIELYRAAN ERIYEEERSL EKILAWTTIF LNKQVQDNSI 201 PDKKLHKLVE FYLRNYKGIT IRLGARRNLE LYDMTYYQAL 241 KSTNRESNLC NEDFLVFARQ DFDIHEAQNQ KGLQQLQRWY 281 ADCRLDTLNF GRDVVIIANY LASLIIGDHA FDYVRLAFAK 321 TSVLVTIMDD FFDCHGSSQE CDKIIELVKE WKENPDAEYG 361 SEELEILFMA LYNTVNELAE RARVEQGRSV KEFLVKLWVE 401 ILSAFKIELD TWSNGTQQSF DEYISSSWLS NGSRLTGLLT 441 MQFVGVKLSD EMLMSEECTD LARHVCMVGR LLNDVCSSER 481 EREENIAGKS YSILLATEKD GRKVSEDEAI AEINEMVEYH 521 WRKVLQIVYK KESILPRRCK DVFLEMAKGT FYAYGINDEL 561 TSPQQSKEDM KSFVF A nucleic acid encoding the Salvia: sclarea sclareol synthase with SEQ ID NO:176 is shown below as SEQ ID NO: 177.

1 ATGTCGCTCG CCTTCAACGT CGGAGTTACG CCTTTCTCCG 41 GCCAAAGAGT TGGGAGCAGG AAAGAAAAAT TTCCAGTCCA 81 AGGATTTCCT GTGACCACCC CCAATAGGTC ACGTCTCATC 121 GTTAACTGCA GCCTTACTAC AATAGATTTC ATGGCGAAAA 161 TGAAAGAGAA TTTCAAGAGG GAAGACGATA AATTTCCAAC 201 GACAACGACT CTTCGATCCG AAGATATACC CTCTAATTTG 241 TGTATAATCG ACACCCTTCA AAGGTTGGGG GTCGATCAAT 231 TCTTCCAATA TGAAATCAAC ACTATTCTAG ATAACACATT 321 CAGGTTGTGG CAAGAAAAAC ACAAAGTTAT ATATGGCAAT 361 GTTACTACTC ATGCAATGGC ATTTAGGCTT TTGCGAGTGA 401 AAGGATACGA AGTTTCATCA GAGGAGTTGG CTCCATATGG 441 TAACCAAGAG GCTGTTAGGC AGCAAACAAA TGACCTGCCG 481 ATGATTATTG AGCTTTATAG AGCAGCAAAT GAGAGAATAT 521 ATGAAGAAGA GAGGAGTCTT GAAAAAATTC TTGCTTGGAC 561 TACCATCTTT CTCAATAAGC AAGTGCAAGA TAACTCAATT 601 CCCGACAAAA AACTGCACAA ACTGGTGGAA TTCTACTTGA 641 GGAATTACAA AGGCATAACC ATAAGATTGG GAGCTAGACG 681 AAACCTCGAG CTATATGACA TGACCTACTA TCAAGCTCTG 721 AAATCTACAA ACAGGTTCTC TAATTTATGC AACGAAGATT 761 TTCTAGTTTT CGCAAAGGAA GATTTCGATA TACATGAAGC 801 CCAGAACCAG AAAGGACTTC AACAACTGCA AAGGTGGTAT 841 GCAGATTGTA GGTTGGACAC CTTAAACTTT GGAAGAGATG 831 TAGTTATTAT TGCTAATTAT TTGGCTTCAT TAATTATTGG 921 TGATCATGCG TTTGACTATG TTCGTCTCGC ATTTGCCAAA 961 ACATCTGTGC TTGTAACAAT TATGGATGAT TTTTTCGACT 1001 GTCATGGCTC TAGTCAAGAG TGTGAGAAGA TCATTGAATT 1041 AGTAAAAGAA TGGAAGGAGA ATCCGGATGC AGAGTACGGA 1081 TCTGAGGAGC TTGAGATCCT TTTTATGGCG TTGTACAATA 1121 CAGTAAATGA GTTGGCGGAG AGGGCTCGTG TTGAACAGGG 1161 GCGTAGTGTC AAAGAGTTTC TAGTCAAACT GTGGGTTGAA 1201 ATACTCTCAG CTTTCAAGAT AGAATTAGAT ACATGGAGCA 1241 ATGGCACGCA GCAAAGCTTC GATGAATACA TTTCTTCGTC 1281 GTGGTTGTCG AACGGTTCCC GGCTGACAGG TCTCCTGACG 1321 ATGCAATTCG TCGGAGTAAA ATTGTCCGAT GAAATGCTTA 1361 TGAGTGAAGA GTGCACTGAT TTGGCTAGGC ATGTCTGTAT 1401 GGTCGGCCGG CTGCTCAACG ACGTGTGCAG TTCTGAGAGG 1441 GAGCGCGAGG AAAATATTGC AGGAAAAAGT TATAGCATTC 1431 TACTAGCAAC TGAGAAAGAT GGAAGAAAAG TTAGTGAAGA 1521 TGAAGCCATT GCAGAGATCA ATGAAATGGT TGAATATCAC 1561 TGGAGAAAAG TGTTGCAGAT TGTGTATAAA AAAGAAAGCA 1601 TTTTGCCAAG AAGATGCAAA GATGTATTTT TGGAGATGGC 1641 TAAGGGTACG TTTTATGCTT ATGGGATCAA CGATGAATTG 1681 ACTTCTCCTC AGCAATCCAA GGAAGATATG AAATCCTTTG 1721 TCTTTTGA

Enzymes described herein can have one or more deletions, insertions, replacements, or substitutions in a part of the enzyme. The enzyme(s) described herein can have, for example, at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 93%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity to a sequence described herein.

In some cases, enzymes can have conservative changes such as one or more deletions, insertions, replacements, or substitutions that have no significant effect on the activities of the enzymes. Examples of conservative substitutions are provided below in Table 1A.

TABLE 1A Conservative Substitutions Type of Amino Acid Substitutable Amino Acids Hydrophilic Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, Thr Sulfhydryl Cys Aliphatic Val, Ile, Leu, Met Basic Lys, Arg, His Aromatic Phe, Tyr, Trp

Due to an increase in resolution at the taxonomic level and consistent clustering of enzymes with identical, or related function, the inventors propose a hierarchical scheme for classifying TPS genes in Lamiaceae from the TPS-e and TPS-c subfamilies. TPS-c genes (class II diTPSs) from Lamiaceae fall broadly into two clades (FIG. 2A), which tire referred to herein as c.1 and c.2. These c.1 and c.2 clades are further divided into three, and two subclades, respectively. The characterized genes from c.1.1 are all ent-CPP [16] synthases, presumably involved in primary metabolism. The taxonomic organization among c.1.1 sequences closely resembles the consensus phytogeny generated from 520 genes from each species (19), together with the short branch lengths compared to other TPS-c clades suggests that diTPSs in c.1.1 are more conserved and evolve more slowly.

The remaining TPS-c clades contain genes involved in specialized metabolism. The only characterized gene from clade c.1.2 is PcTPS1, which makes an ent-labda-8-ene diphosphate product [25]. Enzymes from clade c.1.3 catalyze the production of a variety of products, including ent-CPP [16], ent-8-LPP [7], kolavenyl-PP [36], and 38. 36 and 38 fire the only products without the labdane (Sk4) skeleton produced by Lamiaceae class II diTPSs. Compounds apparently derived from 36 are widespread among Lamiaceae (Table 6), so the inventors hypothesize that the progenitor of c.1.3 was a kolavenyl-PP synthase present in an early common ancestor. The labdane compounds produced by enzymes in c.1 are all in toe ent-configuration. With two exceptions, the known enzymes from clade c.2 all make products with the labdane skeleton in the normal configuration, suggesting that the founder of that clade may have been a normal configuration labdadiene diphosphate synthase. The exceptions are VacTPS3, the only characterized member of c.2.1, which produces syn-CPP [13], and the curious case of SdCPS1, which produces ent-CPP.

Among TPS-e (class I) genes, all but one of the characterized enzymes from e.1 are ent-kaurene [19] synthases, presumably involved in gibberellin biosynthesis. As with the c.1.1 clade, e.1 reflects the taxonomic distribution among the species. Notable in this clade are IrKSL4, which is an ent-atiserene synthase, and SmKSL2, which, in addition to ent-kaurene synthase activity, can convert ent-8-LPP 7 into ent-13-epi-manoyl oxide [6]. Andersen-Ranberg et al, (Angew Chem Int Ed 55(6):2142-2146 (2016)) have tested four of four ent-kaurene synthases and have data indicating that one was from Lamiaceae, which had toe ability to convert 7 to 6, so it is likely that this is a general characteristic of enzymes in the e.1 group.

Most of the specialized class I diTPSs in Lamiaceae fail into clade e.2. Enzymes in e.2 have lost the γ domain, present in many diTPSs, and located on the opposite end of the peptide from the class 1 active site. Characteristic of enzymes in e.2 is their ability to act on multiple substrates. The extreme example is SsSS (Caniard et al. M C Plant Biology 12:119 (2012)) which so far has been able to catalyze the dephosphorylation and minor rearrangement of all class II enzyme products that it has been tested. The range of substrates accepted by other enzymes in this group has not been tested systematically, but among the e.2 enzymes characterized in this study, only one (OmTPS4) accepted ent-CPP, and all accepted (+)-CPP [31], (+)-8-LPP [10], and PgPP [5], There is great diversity toe products of e.2 enzymes, with over 20 distinct compounds represented. Most of toe enzymes in e.2 convert (+)-CPP to miltiradiene [32], and (+)-8-LPP to 13/?-(+)-manoyl oxide [8], with other activities arising sporadically across the clade. Both characterized enzymes in the Nepetoideae specific e.2,2 have unusual activities: IrKSL6 converts (+)-CPP to isopimara-7,15-diene [28], and OmTPS5 converts (+)-CPP to palustradiene [29]. Most of the enzymes in e.2 fall into toe e.2.1 clade which also accounts for most of the known products. Enzymes that we characterized from e.2.1 lent support to emerging functionally consistent subclades. OmTPS4 activity, for three out of four substrates tested, mimics that of its nearest homolog (SsSS), notably accepting ent-CPP as a substrate to produce ent-manool [20]. LITPS4 likewise has activities most similar to its closest homolog, MvELS, converting PgPP into 9,13(S)-epoxy-labd-14-ene [2] with greater specificity than other enzymes tested, although the products from (+)-CPP are different. From the remaining clade, e.2.3, the three characterized enzymes all come from Nepetoideae, and convert (+)-CPP into different products: IrKSL3 produces miltiradiene, IrTPS2 produces nezukol [30], and MsTPS1 produces sandaracopimaradiene [27].

The existence of two strongly supported subclades of specialized diTPSs within c.1, together with the presence of an ent-atiserene synthase in e.1, indicate that the emergence of specialized diTPSs from ent-CPP and ent-kaurene synthases is an ongoing process that has occurred multiple times in the Lamiaceae lineage. While it is evident that candidates selected from anywhere in the phylogenetic tree may have novel activities, clades that seem particularly promising and underexplored are c.2.1, c.1.2, and e.2.3. So far, including this work and previous work, diTPSs have been characterized from only four of the twelve major Lamiaceae clades: Ajugoideae, Lamioideae, Nepetoideae, and Viticoideae. Further expanding to the remaining eight Lamiaceae clades may also be a promising strategy for finding new enzyme activities.

Expression of Enzymes

Also described herein are expression systems that include at least one expression cassette (e.g., expression vectors or transgenes) that encode one or more of the enzyme(s) described herein. The expression systems can also include one or more expression cassettes encoding an enzyme that can synthesize terpene building blocks. For example, the expression systems can include one or more expression cassettes encoding terpene synthases that facilitate production of terpene precursors or building blocks such as those involved in the synthesis of isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP).

Cells containing such expression systems are further described herein. The cells containing such expression systems can be used to manufacture the enzymes (e.g., for in vitro use) and/or one or more terpenes, diterpenes, or terpenoids produced by the enzymes. Methods of using the enzymes or cells containing expression cassettes encoding such enzymes to make products such as terpenes, diterpenes, terpenoids, and combinations thereof are also described herein.

Nucleic acids encoding the enzymes can have sequence modifications. For example, nucleic acid sequences described herein can be modified to express enzymes that have modifications. Most amino acids can be encoded by more than one codon. When an amino acid is encoded by more than one codon, the codons are referred to as degenerate codons. A listing of degenerate codons is provided in Table 1B below.

TABLE 1B Degenerate Amino Acid Codons Amino Acid Three Nucleotide Codon Ala/A GCT, GCC, GCA, GCG Arg/R CGT, CGC, CGA, CGG, AGA, AGG Asn/N AAT, AAC Asp/D GAT, GAC Cys/C TGT, TGC Gln/Q CAA, CAG Glu/E GAA, GAG Gly/G GGT, GGC, GGA, GGG His/H CAT, CAC Ile/I ATT, ATC, ATA Leu/L TTA, TTG, CTT, CTC, CTA, CTG Lys/K AAA, AAG Met/M ATG Phe/F TTT, TTC Pro/P CCT, CCC, CCA, CCG Ser/S TCT, TCC, TCA, TCG, AGT, AGC Thr/T ACT, ACC, ACA, ACG Trp/W TGG Tyr/Y TAT, TAC Val/V GTT, GTC, GTA, GTG START ATG STOP TAG, TGA, TAA

Different organisms may translate different codons more or less efficiently (e.g., because they have different ratios of tRNAs) than other organisms. Hence, when some amino acids can be encoded by several codons, a nucleic acid segment can be designed to optimize the efficiency of expression of an enzyme by using codons that are preferred by an organism, of interest. For example, the nucleotide coding regions of the enzymes described herein can be codon optimized for expression in various plant species. For example, many of the enzymes described herein were originally isolated from the mint family (Lamiaceae), however such enzymes can be expressed in a variety of host cells, including for example, as Nicotiana benthamiana, Nicotiana tabacum, Nicotiana rustica, Nicotiana excelsior, and Nicotiana excelsiana.

An optimized nucleic acid can have less than 98%, less than 97%, less than 95%, or less than 94%, or less than 93%, or less than 92%, or less than 91%, or less than 90%, or less than 89%, or less than 88%, or less than 85%, or less than 83%, or less than 80%, or less than 75% nucleic acid sequence identity to a corresponding non-optimized (e.g., a non-optimized parental or wild type enzyme nucleic acid) sequence.

The enzymes described herein can be expressed from an expression cassette and/or an expression vector. Such an expression cassette can include a nucleic acid segment that encodes an enzyme operably linked to a promoter to drive expression of the enzyme. Convenient vectors, or expression systems can be used to express such enzymes, in some instances, the nucleic acid segment encoding an enzyme is operably linked to a promoter and/or a transcription termination sequence. The promoter and/or the termination sequence can be heterologous to the nucleic acid segment that encodes an enzyme. Expression cassettes can have a promoter operably linked to a heterologous open reading frame encoding an enzyme. The invention therefore provides expression cassettes or vectors useful for expressing one or more enzyme(s).

Constructs, e.g., expression cassettes, and vectors comprising the isolated nucleic acid molecule, e.g., with optimized nucleic acid sequence, as well as kits comprising the isolated nucleic acid molecule, construct or vector are also provided.

The nucleic acids described herein can also be modified to improve or alter toe functional properties of the encoded enzymes. Deletions, insertions, or substitutions can be generated by a variety of methods such as, but not limited to, random mutagenesis and/or site-specific recombination-mediated methods. The mutations can range in size from one or two nucleotides to hundreds of nucleotides (or any value there between). Deletions, insertions, and/or substitutions are created at a desired location in a nucleic acid encoding the enzyme(s).

Nucleic acids encoding one or more enzyme(s) can have one or more nucleotide deletions, insertions, replacements, or substitutions. For example, the nucleic acids encoding one or more enzyme(s) can, for example, have less than 95%, or less than 94.8%, or less than 94.5%, or less than 94%, or less than 93.8%, or less than 94.50% nucleic acid sequence identity to a corresponding parental or wild-type sequence. In some cases, the nucleic acids encoding one or more enzyme(s) can have, for example, at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at 90% sequence identity to a corresponding parental or wild-type sequence. Examples of parental or wild type nucleic acid sequences for unmodified enzyme(s) with amino acid sequences SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 57, 59, or 176 include nucleic acid sequences SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, or 177 respectively. Any of these nuclei acid or amino acid sequences can, for example, encode or have enzyme sequences with less than 99%, less than 98%, less than 97%, less than 96%, less than 95%, less than 94.8%, less than 94.5%, less than 94%, less than 93.8%, less than 93.5%, less than 93%, less than 92%, less than 91%, or less than 90% sequence identity to a corresponding parental or wild-type sequence.

Also provided are nucleic acid molecules (polynucleotide molecules) that can include a nucleic acid segment encoding an enzyme with a sequence that is optimized for expression in at least one selected host organism or host cell. Optimized sequences include sequences which are codon optimized, i.e., codons which are employed more frequently in one organism relative to another organism. In some cases, the balance of codon usage is such that the most frequently used codon is not used to exhaustion. Other modifications can include addition or modification of Kozak sequences and/or moons, and/or to remove undesirable sequences, for instance, potential transcription factor binding sites.

An enzyme useful tor synthesis of terpenes, diterpenes, and terpenoids may be expressed on the surface of, or within, a prokaryotic or eukaryotic cell. In some cases, expressed enzyme(s) can be secreted by that ceil.

Techniques of molecular biology, microbiology, and recombinant DNA technology which are within the skill of the art can be employed to make and use the enzymes, expression systems, and terpene products described herein. Such techniques available in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989); DNA Cloning, Vols. I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Animal Cell Culture (R. K. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL press, 1986); Perbal, B., A Practiced Guide to Molecular Cloning (1984); the series Methods In Enzymology (S. Colowick and N, Kaplan eds., Academic Press, Inc.); Current Protocols In Molecular Biology (John Wiley & Sons, Inc), Current Protocols In Protein Science (John Wiley & Sons, Inc), Current Protocols In Microbiology (John Wiley & Sons, Inc), Current Protocols In Nucleic Acid Chemistry (John Wiley & Sons, Inc), and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds., 1986, Blackwell Scientific Publications).

Modified plants that contain nucleic acids encoding enzymes within their somatic and/or germ cells are described herein. Such genetic modification can be accomplished by available procedures. For example, one of skill in the art can prepare an expression cassette or expression vector that can express one or more encoded enzymes. Plant cells can be transformed by the expression cassette or expression vector, and whole plants (and their seeds) can be generated from the plant cells that were successfully transformed with the enzyme nucleic acids. Some procedures for making such genetically modified plants and their seeds are described below.

Promoters: The nucleic acids encoding enzymes can be operably linked to a promoter, which provides for expression of mRNA from the nucleic acids encoding the enzymes. The promoter is typically a promoter functional in plants and can be a promoter functional during plant growth and development. A nucleic acid segment encoding an enzyme is operably linked to the promoter when it is located downstream from the promoter. The combination of a coding region for an enzyme operably linked to a promoter forms an expression cassette, which can optionally include other elements as well.

Promoter regions are typically found in the flanking DNA upstream from the coding sequence in both the prokaryotic and eukaryotic cells. A promoter sequence provides for regulation of transcription of the downstream gene sequence and typically includes from about 50 to about 2,000 nucleotide base pairs. Promoter sequences also contain regulatory sequences such as enhancer sequences that can influence the level of gene expression. Some isolated promoter sequences can provide for gene expression of heterologous DNAs, that is a DNA different from the native or homologous DNA.

Promoter sequences are also known to be strong or weak, or inducible. A strong promoter provides for a high level of gene expression, whereas a weak promoter provides for a very low level of gene expression. An inducible promoter is a promoter that provides for the turning gene expression on and off in response to an exogenously added agent, or to an environmental or developmental stimulus. For example, a bacterial promoter such as the P_(tac) promoter can be induced to varying levels of gene expression depending on the level of isopropyl-beta-D-thiogalactoside added to the transformed cells.

Promoters can also provide for tissue specific or developmental regulation. An isolated promoter sequence that is a strong promoter for heterologous DNAs is advantageous because it provides for a sufficient level of gene expression for easy detection and selection of transformed cells and provides for a high level of gene expression when desired.

Expression cassettes generally include, but tire not limited to, examples of plant promoters such as the CaMV 35S promoter (Odell et al., Nature. 313:810-812 (1985)), or others such as CaMV 19S (Lawton et al., Plant Molecular Biology. 9:315-324 (1987)), nos (Ebert et al., Proc. Natl. Acad. Sci. USA. 84:5745-5749 (1987)), AdhI (Walker et al., Proc. Natl. Acad. Sci. USA. 84:6624-6628 (1987)), sucrose synthase (Yang et al., Proc. Natl. Acad. Sci. USA. 87:4144-4148 (1990)), α-tubulin, ubiquitin, actin (Wang et al., Mol. Cell. Biol. 12:3399 (1992)), cab (Sullivan et al, Mol Gen. Genet. 215:431 (1989)), PEPCase (Hudspeth et al., Plant Molecular Biology. 12:579-589 (1989)) or those associated with the R gene complex (Chandler et al., The Plant Cell. 1:1175-1183 (1989)). Further suitable promoters include a CYP71D16 trichome-specific promoter and the CBTS (cembratrienol synthase) promotor, cauliflower mosaic virus promoter, the Z10 promoter from a gene encoding a 10 kD zein protein, a Z27 promoter from a gene encoding a 27 kD zein protein, the plastid rRNA-operon (rrn) promoter, inducible promoters, such as the light inducible promoter derived from the pea rbcS gene (Coruzzi et al., EMBO J. 3:1671 (1971)), RUBISCO-SSU light inducible promoter (SSU) from tobacco and the actin promoter from rice (McElroy et al., The Plant Cell. 2:163-171 (1990)). Other promoters that are useful can also be employed.

Alternatively, novel tissue specific promoter sequences may be employed. cDNA clones from a particular tissue can be isolated and those clones which are expressed specifically in that tissue can be identified, for example, using Northern blotting. Preferably, the gene isolated is not present in a high copy number but is relatively abundant in specific tissues. The promoter and control elements of corresponding genomic clones can then be localized using techniques well known to those of skill in the art.

A nucleic acid encoding an enzyme can be combined with the promoter by standard methods to yield an expression cassette, for example, as described in Sambrook et al. (MOLECULAR CLONING: A LABORATORY MANUAL. Second Edition (Cold Spring Harbor, N.Y.: Cold Spring Harbor Press (1989); MOLECULAR CLONING: A LABORATORY MANUAL. Third Edition (Cold Spring Harbor, N.Y.: Cold Spring Harbor Press (2000)). Briefly, a plasmid containing a promoter such as the 35S CaMV promoter or the CYP71D16 trichome-specific promoter can be constructed as described in Jefferson (Plant Molecular Biology Reporter 5:387-405 (1987)) or obtained from Clontech Lab in Palo Alto, Calif. (e.g., pBI121 or pBI221). Typically, these plasmids are constructed to have multiple cloning sites having specificity for different restriction enzymes downstream from the promoter.

The nucleic acid sequence encoding for the enzyme(s) can be subcloned downstream from the promoter using restriction enzymes and positioned to ensure that the DNA is inserted in proper orientation with respect to the promoter so that the DNA can be expressed as sense RNA. Once the nucleic acid segment encoding the enzyme is operably linked to a promoter, the expression cassette so formed can be subcloned into a plasmid or other vector (e.g., an expression vector).

In some embodiments, a cDNA clone encoding an enzyme is isolated from a mint species, for example, from leaf, trichome, or root tissue. In other embodiments, cDNA clones from other species (that encode an enzyme) are isolated from, selected plant tissues, or a nucleic acid encoding a wild type, mutant or modified enzyme is prepared by available methods or as described herein. For example, the nucleic acid encoding the enzyme can be any nucleic acid with a coding region that hybridizes to SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, or 177, and that has enzyme activity. Using restriction endonucleases, the entire coding sequence for the enzyme is subcloned downstream of the promoter in a 5′ to 3′ sense orientation.

Targeting Sequences: Additionally, expression cassettes can be constructed and employed to target the nucleic acids encoding an enzyme to an intracellular compartment within plant cells or to direct an encoded protein to the extracellular environment. This can generally be achieved by joining a DNA sequence encoding a transit or signal peptide sequence to the coding sequence of the nucleic acid encoding the enzyme. The resultant transit, or signal, peptide can transport the protein to a particular intracellular, or extracellular, destination and can then be co-translationally or post-translationally removed. Transit peptides act by facilitating the transport of proteins through intracellular membranes, e.g., vacuole, vesicle, plastid and mitochondrial membranes, whereas signal peptides direct proteins through the extracellular membrane. By facilitating transport of the protein into compartments inside or outside the cell, these sequences can increase the accumulation of a particular gene product within a particular location. For example, see U.S. Pat. No. 5,258,300.

For example, in some cases it may be desirable to localize the enzymes to the plastidic compartment and/or within plant cell trichomes. The best compliment of transit peptides/secretion peptide/signal peptides can be empirically ascertained. The choices can range from using the native secretion signals akin to the enzyme candidates to be transgenically expressed, to transit peptides from proteins known to be localized into plant organelles such as trichome plastid s in general. For example, transit peptides can be selected from proteins that have a relative high titer in the trichomes. Examples include, but not limited to, transit peptides form a terpenoid cyclase (e.g. cembratrieneol cyclase), the LTP1 protein, the Chlorophyll a-b binding protein 40, Phylloplanin, Glycine-rich Protein (GRP), Cytochrome P450 (CYP71D16); all from Nicotiana sp. alongside RUBISCO (Ribulose bisphosphate carboxylase) small unit protein from both Arabidopsis and Nicotiana sp.

3′ Sequences: When the expression cassette is to be introduced into a plant ceil, the expression cassette can also optionally include 3′ untranslated plant regulatory DNA sequences that act as a signal to terminate transcription and allow for the polyadenylation of the resultant mRNA. The 3′ untranslated regulatory DNA sequence can include from about 300 to 1,000 nucleotide base pairs and can contain plant transcriptional and translational termination sequences. For example, 3′ elements that can be used include those derived from the nopaline synthase gene of Agrobacterium tumefaciens (Bevan et al., Nucleic Acid Research. 11:369-385 (1983)), or the terminator sequences for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens, and/or the 3′ end of the protease inhibitor I or II genes from potato or tomato. Other 3′ elements known to those of skill in the art can also be employed. These 3′ untranslated regulatory sequences can be obtained as described in An (Methods in Enzymology. 153:292 (1987)). Many such 3′ untranslated regulatory sequences are already present in plasmids available from commercial sources such as Clontech, Palo Alto, Calif. The 3′ untranslated regulatory sequences can be operably linked to the 3′ terminus of the nucleic acids encoding the enzyme.

Selectable and Screenable Marker Sequences: To improve identification of transformants, a selectable or screenable marker gene can be employed with the expressible nucleic acids encoding the enzyme(s). “Marker genes” are genes that impart a distinct phenotype to cells expressing the marker gene and thus allow such transformed cells to be distinguished from cells that do not have the marker. Such genes may encode either a selectable or a screenable marker, depending on whether the marker confers a trait which one can ‘select’ for by chemical means, i.e., through the use of a selective agent (e.g., a herbicide, antibiotic, or the like), or whether it is simply a trait that one can identify through observation or testing, i.e., by ‘screening’ (e.g., foe R-locus trait). Of course, many examples of suitable marker genes are available can be employed in the practice of the invention.

Included within the terms ‘selectable or screenable marker genes’ are also genes which encode a “secretable marker” whose secretion can be detected as a means of identifying or selecting for transformed cells. Examples include markers which encode a secretable antigen that can be identified by antibody interaction, or secretable enzymes that can be detected by their catalytic activity. Secretable proteins fall into a number of classes, including small, diffusible proteins detectable, e.g., by ELISA; and proteins that are inserted or trapped in the cell wall (e.g., proteins that include a leader sequence such as that found in the expression unit of extensin or tobacco PR-S).

With regard to selectable secretable markers, the use of an expression system that encodes a polypeptide that becomes sequestered in the cell wall, where the polypeptide includes a unique epitope may be advantageous. Such a cell wall antigen can employ an epitope sequence that would provide low background in plant tissue, a promoter-leader sequence that imparts efficient expression and targeting across the plasma membrane, and that can produce protein that is bound in the cell wall and yet is accessible to antibodies. A normally secreted ceil wall protein modified to include a unique epitope would satisfy such requirements.

Example of protein markers suitable for modification in this manner include extensin or hydroxyproline rich glycoprotein (HPRG), For example, the maize HPRG (Stiefel et al., The Plant Cell. 2:785-793 (1990)) is well characterized in terms of molecular biology, expression, and protein structure and therefore can readily be employed. However, any one of a variety of extensins and/or glycine-rich ceil wall proteins (Keller et al, EMBO J. 8:1309-1314 (1989)) could be modified by the addition of an antigenic site to create a screenable marker.

Selectable markers for use in connection with the present invention can include, but are not limited to, a neo gene (Potrykus et al., Mol. Gen. Genet. 199:183-188 (1985)) which codes for kanamycin resistance and can be selected for using kanamycin, G418; a bar gene which codes for bialaphos resistance; a gene which encodes an altered EPSP synthase protein (Hinchee et al., Bio/Technology. 6:915-922 (1988)) thus conferring glyphosate resistance; a nitrilase gene such as bxn from Klebsiella azaenae which confers resistance to bromoxynil (Stalker et al., Science. 242:419-423 (1988)); a mutant acetolactate synthase gene (ALS) which confers resistance to imidazolinone, sulfonylurea or oilier ALS-inhibiting chemicals (European Patent Application 154, 204 (1985)); a methotrexate-resistant DHFK gene (Thiliet et al., J. Biol. Chem. 263:12500-12508 (1988)); adalapon dehalogenase gene that confers resistance to the herbicide dalapon; or a mutated anthranilate synthase gene that confers resistance to 5-methyl tryptophan. Where a mutant EPSP synthase gene is employed, additional benefit may be realized through the incorporation of a suitable chloroplast transit peptide, CTP (European Patent Application 0 218 571 (1987)).

An illustrative embodiment of a selectable marker gene capable of being used in systems to select transformants is the gene that encode the enzyme phosphinothricin acetyltransferase, such as the bar gene from Streptomyces hygroscopicus or the pat gene from Streptomyces viridochromogenes (U.S. Pat. No. 5,550,318). The enzyme phosphinothricin acetyl transferase (PAT) inactivates the active ingredient in the herbicide bialaphos, phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami et al., Mol. Gen. Genet. 205:42-50 (1986); Twell et al., Plant Physiol. 91:1270-1274 (1989)) causing rapid accumulation of ammonia and cell death. Screenable markers that may be employed include, hut are not limited to, a β-glucuronidase or uidA gene (GUS) that encodes an enzyme for which various chromogenic substrates are known; an R-locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al., In: Chromosome Structure and Function: Impact of New Concepts, 18^(th) Stabler Genetics Symposium, J. P. Gustafson and R. Appels, eds. (New York: Plenum Press) pp. 263-282 (1988)); a β-lactamase gene (Sutcliffe, Proc. Natl Acad. Sci. USA. 75:3737-3741 (1978)), which encodes an enzyme for which various chromogenic substrates are known (e.g., PADAC, a chromogenic cephalosporin); a xy/E gene (Zukowsky et al., Proc. Natl. Acad. Sci. USA. 80:1101 (1983)) which encodes a catechol dioxygenase that can convert chromogenic catechols; an α-amylase gene (Ikuta et al., Bio/technology 8:241-242 (1990)); a tyrosinase gene (Katz et al., J. Gen. Microbiol. 129:2703-2714 (1983)) which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone which in turn condenses to form the easily detectable compound melanin; a β-galactosidase gene, which encodes an enzyme for which there are chromogenic substrates; a luciferase (lux) gene (Ow et al., Science. 234:856-859.1986), which allows for bioluminescence detection; or an aequorin gene (Prasher et al., Biochem. Biophys. Res. Comm. 126:1259-1268 (1985)), which may be employed in calcium-sensitive bioluminescence detection, or a green or yellow fluorescent protein gene (Niedz et ah, Plant Cell Reports. 14:403 (1995)).

Another screenable marker contemplated for use is firefly luciferase, encoded by the lux gene. The presence of the lux gene in transformed cells may be detected using, for example, X-ray film, scintillation counting, fluorescent spectrophotometry, low-light video cameras, photon counting cameras or multiwell luminometry. It is also envisioned that this system may be developed for population screening for bioluminescence, such as on tissue culture plates, or even for whole plant screening.

Other Optional Sequences: An expression cassette of the invention can also include plasmid DMA. Plasmid vectors include additional DNA sequences that provide for easy selection, amplification, and transformation of the expression cassette in prokaryotic and eukaryotic cells, e.g., pUC-derived vectors such as pUC8, pUC9, pUC18, pUC19, pUC23, pUC119, and pUC120, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors, or pBS-derived vectors. The additional DNA sequences can include origins of replication to provide for autonomous replication of the vector, additional selectable marker genes, for example, encoding antibiotic or herbicide resistance, unique multiple cloning sites providing for multiple sites to insert DNA sequences or genes encoded in the expression cassette and sequences that enhance transformation of prokaryotic and eukaryotic cells.

Another vector that is useful for expression in both plant and prokaryotic cells is the binary Ti plasmid (as disclosed in Schilperoort et al., U.S. Pat. No. 4,940,838) as exemplified by vector pGA582. This binary Ti plasmid vector has been previously characterized by An (Methods in Enzymology. 153:292 (1987)) and is available from Dr. An. This binary Ti vector can be replicated in prokaryotic bacteria such as E. coli and Agrobacterium. The Agrobacterium plasmid vectors can be used to transfer the expression cassette to dicot plant cells, and under certain conditions to monocot cells, such as rice cells. The binary Ti vectors can include the nopaline T DNA right and left borders to provide for efficient plant cell transformation, a selectable marker gene, unique multiple cloning sites in the T border regions, the cold replication of origin and a wide host range replicon. The binary Ti vectors carrying an expression cassette of the invention can be used to transform both prokaryotic and eukaryotic cells but is usually used to transform dicot plant cells.

DNA Delivery of the DNA Molecules into Host Cells: Methods described herein can include introducing nucleic acids encoding enzymes, such as a preselected cDNA encoding the selected enzyme, into a recipient cell to create a transformed cell. In some instances, the frequency of occurrence of cells taking up exogenous (foreign) DNA may be low. Moreover, it is most likely that not all recipient cells receiving DNA segments or sequences will result in a transformed cell wherein the DNA is stably integrated into the plant genome and/or expressed. Some recipient cells may show only initial and transient gene expression. However, certain cells from virtually any dicot or monocot species may be stably transformed, and these cells regenerated into transgenic plants, through tire application of the techniques disclosed herein.

Another aspect of the invention is a plant that can produce terpenes, diterpenes and terpenoids, wherein the plant has introduced nucleic acid sequence(s) encoding one or more enzymes. The plant can be a monocotyledon or a dicotyledon. Another aspect of the invention includes plant cells (e.g., embryonic cells or other cell lines) that can regenerate fertile transgenic plants and/or seeds. The cells can be derived from either monocotyledons or dicotyledons, hi some embodiments, the plant or cell is a monocotyledon plant or cell. In some embodiments, the plant or cell is a dicotyledon plant or cell. For example, the plant or cell can be a tobacco plant or cell. The cell(s) may be in a suspension cell culture or may be in an intact plant part, such as an immature embryo, or in a specialized plant tissue, such as callus, such as Type I or Type II callus.

Transformation of plant cells can be conducted by any one of a number of methods available in the art. Examples are: Transformation by direct DNA transfer into plant cells by electroporation (U.S. Pat. Nos. 5,384,253 and 5,472,869, Dekeyser et al., The Plant Cell. 2:591-602 (1990)); direct DNA transfer to plant cells by PEG precipitation (Hayashimoto et al., Plant Physiol. 93:857-863 (1990)); direct DNA transfer to plant cells by microprojectile bombardment (McCabe et al., Bio/Technology. 6:923-926 (1988); Gordon-Kamm et al, The Plant Cell. 2:603-618 (1990); U.S. Pat. Nos. 5,489,520; 5,538,877; and 5,538,880) and DNA transfer to plant cells via infection with Agrobacterium. Methods such as microprojectile bombardment or electroporation can be carried out with “naked” DNA where the expression cassette may be simply carried on any E. coli-derived plasmid cloning vector. In the case of viral vectors, it is desirable that the system retain replication functions, but lack the functions tor disease induction.

One method for dicot transformation, for example, involves infection of plant ceils with Agrobacterium tumefaciens using the leaf-disk protocol (Horsch et al., Science 227:1229-1231 (1985). Methods for transformation of monocotyledonous plants utilizing Agrobacterium tumefaciens have been described by Hiei et al. (European Patent 0 604 662, 1994) and Saito et al. (European Patent 0 672 752, 1995).

Monocot cells such as various grasses or dicot cells such as tobacco can be transformed via microprojectile bombardment of embryogenic callus tissue or immature embryos, or by electroporation following partial enzymatic degradation of the cell wall with a pectinase-containing enzyme (U.S. Pat. Nos. 5,384,253; and 5,472,869). For example, embryogenic cell lines derived from immature embryos can be transformed by accelerated particle treatment as described by Gordon-Kamm et al. (The Plant Cell. 2:603-618 (1990)) or U.S. Pat. Nos. 5,489,520; 5,538,877 and 5,538,880, cited above. Excised immature embryos can also be used as the target for transformation prior to tissue culture induction, selection and regeneration as described in U.S. application Ser. No. 08/112,245 and PCT publication WO 95/06128.

The choice of plant tissue source for transformation may depend on the nature of the host plant and the transformation protocol. Useful tissue sources include callus, suspensions culture cells, protoplasts, leaf segments, stem segments, tassels, pollen, embryos, hypocotyls, tuber segments, meristematic regions, and the like. The tissue source is selected and transformed so that it retains the ability to regenerate whole, fertile plants following transformation, i.e., contains totipotent ceils.

The transformation is carried out under conditions directed to the plant tissue of choice. The plant cells or tissue are exposed to the DNA or RNA encoding enzymes for an effective period of time. This may range from a less than one second pulse of electricity for electroporation to a 2-day to 3-day co-cultivation in the presence of plasmid-bearing Agrobacterium cells. Buffers and media used will also vary with the plant tissue source and transformation protocol. Many transformation protocols employ a feeder layer of suspended culture cells (tobacco, for example) on the surface of solid media plates, separated by a sterile filter paper disk from the plant cells or tissues being transformed.

Electroporation: Where one wishes to introduce DNA by means of electroporation, it is contemplated that the method of Krzyzek et al. (U.S. Pat. No. 5,384,253) may be advantageous. In this method, certain cell wall-degrading enzymes, such as pectin-degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells. Alternatively, recipient cells can be made more susceptible to transformation, by mechanical wounding.

To effect transformation by electroporation, one may employ either friable tissues such as a suspension cell cultures, or embryogenic callus, or alternatively, one may transform immature embryos or other organized tissues directly. The cell walls of the preselected cells or organs can be partially degraded by exposing them to pectin-degrading enzymes (pectinases or pectolyases) or mechanically wounding them in a controlled manner. Such cells would then be receptive to DNA uptake by electroporation, which may be carried out at tins stage, and transformed cells then identified by a suitable selection or screening protocol dependent on the nature of the newly incorporated DNA.

Microprojectile Bombardment: A further advantageous method for delivering transforming DNA segments to plant cells is microprojectile bombardment. In this method, microparticles may be coated with DNA and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, gold, platinum, and the like.

It is contemplated that in some instances DNA precipitation onto metal particles would not be necessary for DNA delivery to a recipient cell using microprojectile bombardment. In an illustrative embodiment, non-embryogenic BMS cells were bombarded with intact cells of the bacteria E. coli or Agrobacterium, tumefaciens containing plasmids with either the β-glucoronidase or bar gene engineered for expression in selected plant cells. Bacteria were inactivated by ethanol dehydration prior to bombardment. A low level of transient expression of the β-glucoronidase gene was observed 24-48 hours following DNA delivery. In addition, stable transformants containing the bar gene were recovered following bombardment with either E. coli or Agrobacterium tumefaciens cells. It is contemplated that particles may contain DNA rather than be coated with DNA. Hence it is proposed that particles may increase the level of DNA delivery but are not, in and of themselves, necessary to introduce DNA into plant cells.

An advantage of microprojectile bombardment, in addition to being an effective means of reproducibly stably transforming monocots, microprojectile bombardment does not require the isolation of protoplasts (Christou et al., PNAS 84:3962-3966 (1987)), the formation of partially degraded cells, and no susceptibility to Agrobacterium infection is required. An illustrative embodiment of a method for delivering DNA into maize cells by acceleration is a Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with maize cells cultured in suspension (Gordon-Kamm et al, The Plant Cell. 2:603-618 (1990)). The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. It is believed that a screen intervening between the projectile apparatus and the cells to be bombarded reduces the size of projectile aggregate and may contribute to a higher frequency of transformation, by reducing the damage inflicted on recipient cells by an aggregated projectile.

For bombardment, cells in suspension are preferably concentrated on filters or solid culture medium. Alternatively, immature embryos or other target cells may be arranged on solid culture medium. The ceils to be bombarded are positioned at an appropriate distance below the microprojectile stopping plate. If desired, one or more screens are also positioned between the acceleration device and the cells to be bombarded. Through the use of techniques set forth herein, one may obtain up to 1000 or more foci of cells transiently expressing a marker gene. The number of cells in a focus which express the exogenous gene product 48 hours post-bombardment often range from about 1 to 10 and average about 1 to 3.

In bombardment transformation, one may optimize the prebombardment culturing conditions and the bombardment parameters to yield the maximum numbers of stable transformants. Both the physical and biological parameters for bombardment can influence transformation frequency. Physical factors are those that involve manipulating the DNA/microprojectile precipitate or those that affect the path and velocity of either the macro- or microprojectiles. Biological factors include all steps involved in manipulation of cells before and immediately after bombardment, the osmotic adjustment of target cells to help alleviate the trauma associated with the bombardment, and also the nature of the transforming DNA, such as linearized DNA or intact supercoiled plasmid DNA.

One may wish to adjust various bombardment parameters in small scale studies to fully optimize the conditions and/or to adjust physical parameters such as gap distance, flight distance, tissue distance, and helium pressure. One may also minimize the trauma reduction factors (TRFs) by modifying conditions which influence the physiological state of the recipient cells and which may therefore, influence transformation and integration efficiencies. For example, tire osmotic state, tissue hydration and the subculture stage or cell cycle of the recipient cells may be adjusted for optimum transformation. Execution of such routine adjustments will be known to those of skill in the art.

Selection: An exemplary embodiment of methods for identifying transformed cells involves exposing the bombarded cultures to a selective agent, such as a metabolic inhibitor, an antibiotic, or the like. Cells which have been transformed and have stably integrated a marker gene conferring resistance to the selective agent used, will grow and divide in culture. Sensitive ceils will not be amenable to further culturing.

To use the bar-bialaphos or the EPSPS-glyphosate selective system, bombarded tissue is cultured for about 0-28 days on nonselective medium and subsequently transferred to medium containing from about 1-3 mg/l bialaphos or about 1-3 mM glyphosate, as appropriate. While ranges of about 1-3 mg/l bialaphos or about 1-3 mM glyphosate can be employed, it is proposed that ranges of at least about 0.1-50 mg/l bialaphos or at least about 0.1-50 mM glyphosate will find utility in the practice of the invention. Tissue can be placed on any porous, inert, solid or semi-solid support for bombardment, including but not limited to filters and solid culture medium. Bialaphos and glyphosate are provided as examples of agents suitable for selection of transformants, but the technique of this invention is not limited to them.

The enzyme luciferase is also useful as a screenable marker in the context of the present invention. In the presence of the substrate luciferin, ceils expressing luciferase emit light which can be detected on photographic or X-ray film, in a luminometer for liquid scintillation counter), by devices that enhance night vision, or by a highly light sensitive video camera, such as a photon counting camera. All of these assays are nondestructive and transformed cells may be cultured further following identification. The photon counting camera is especially valuable as it allows one to identify specific cells or groups of cells which are expressing luciferase and manipulate those in real time.

It is further contemplated that combinations of screenable and selectable markers may be useful for identification of transformed cells. For example, selection with a growth inhibiting compound, such as bialaphos or glyphosate at concentrations that provide 100% inhibition followed by screening of growing tissue for expression of a screenable marker gene such as luciferase would allow one to recover transformants from, cell or tissue types that are not amenable to selection alone.

Regeneration and Seed Production: Cells that survive the exposure to the selective agent, or cells that have been scored positive in a screening assay, are cultured in media that supports regeneration of plants. One example of a growth regulator that can be used for such purposes is dicamba or 2,4-D. However, other growth regulators may be employed, including NAA, NAA+2,4-D or perhaps even picloram. Media improvement in these and like ways can facilitate the growth of cells at specific developmental stages. Tissue can be maintained on a basic media with growth regulators until sufficient tissue is available to begin plant regeneration efforts, or following repeated rounds of manual selection, until the morphology of the tissue is suitable for regeneration, at least two weeks, then transferred to media conducive to maturation of embryoids. Cultures are typically transferred every two weeks on this medium. Shoot development signals the time to transfer to medium lacking growth regulators.

The transformed cells, identified by selection or screening and cultured in an appropriate medium that supports regeneration, can then be allowed to mature into plants. Developing plantlets are transferred to soilless plant growth mix, and hardened, e.g., in an environmentally controlled chamber at about 85% relative humidity, about 600 ppm CO₂, and at about 25-250 microeinsteins/sec·m² of light. Plants can be matured either in a growth chamber or greenhouse. Plants are regenerated from about 6 weeks to 10 months after a transformant is identified, depending on the initial tissue. During regeneration, cells are grown on solid media in tissue culture vessels. Illustrative embodiments of such vessels are petri dishes and Plant Con™. Regenerating plants can be grown at about 19° C. to 28° C. After the regenerating plants have reached the stage of shoot and root development, they may be transferred to a greenhouse for further growth and testing.

Mature plants are then obtained from cell lines that are known to express the trait. In some embodiments, the regenerated plants are self-pollinated. In addition, pollen obtained from the regenerated plants can be crossed to seed grown plants of agronomically important inbred lines. In some cases, pollen from plants of these inbred lines is used to pollinate regenerated plants. The trait is genetically characterized by evaluating the segregation of the trait in first and later generation progeny. The heritability and expression in plants of traits selected in tissue culture are of particular importance if the traits fire to be commercially useful.

Regenerated plants can be repeatedly crossed to inbred plants to introgress the nucleic acids encoding an enzyme into the genome of the inbred plants. This process is referred to as backcross conversion. When a sufficient number of crosses to the recurrent inbred parent have been completed in order to produce a product of the backcross conversion process that is substantially isogenic with the recurrent inbred parent except for the presence of the introduced nucleic acids, the plant is self-pollinated at least once in order to produce a homozygous backcross converted inbred containing the nucleic acids encoding the enzyme(s). Progeny of these plants are true breeding.

Alternatively, seed from transformed plants regenerated from transformed tissue cultures is grown in the field and self-pollinated to generate hue breeding plants.

Seed from the fertile transgenic plants can then be evaluated for the presence and/or expression of the enzyme(s). Transgenic plant and/or seed tissue can be analyzed for enzyme expression using methods such as SDS polyacrylamide gel electrophoresis, Western blot, liquid chromatography (e.g., HPLC) or other means of detecting an enzyme product (e.g., a terpene, diterpene, terpenoid, or a combination thereof).

Once a transgenic seed expressing the enzyme(s) and producing one or more terpenes, diterpenes, and/or terpenoids in the plant is identified, the seed can be used to develop true breeding plants. The true breeding plants are used to develop a line of plants expressing terpenes, diterpenes, and/or terpenoids in various plant tissues (e.g., in leaves, bracts, and/or trichomes) while still maintaining other desirable functional agronomic traits. Adding the trait of terpene, diterpene, and/or terpenoid production can be accomplished by back-crossing with selected desirable functional agronomic trait(s) and with plants that do not exhibit such traits and studying the pattern of inheritance in segregating generations. Those plants expressing the target trait(s) in a dominant fashion are preferably selected. Back-crossing is canned out by crossing the original fertile transgenic plants with a plant from an inbred line exhibiting desirable functional agronomic characteristics while not necessarily expressing the trait of terpene, diterpene, and/or terpenoid production in the plant. The resulting progeny can then be crossed back to the parent that expresses the terpenes, diterpenes, and/or terpenoids. The progeny from this cross will also segregate so that some of the progeny carry the trait and some do not. This back-crossing is repeated until the goal of acquiring an inbred line with the desirable functional agronomic traits, and with production of terpenes, diterpenes, and/or terpenoids within various tissues of the plant is achieved. The enzymes can be expressed in a dominant fashion.

Subsequent to back-crossing, the new transgenic plants can be evaluated for synthesis of terpenes, diterpenes, and/or terpenoids in selected plant lines. This can be done, for example, by gas chromatography, mass spectroscopy, or NMR analysis of whole plant cell walls (Kim, H., and Ralph, I. Solution-state 2D NMR of ball-milled plant cell wall gels in DMSO-d₆/pyridine-d₅. (2010) Org. Bio mol. Chem. 8(3), 576-591; Yelie, D. J., Ralph, J., and Frihart, C. R. Characterization of non-derivatized plant cell walls using high-resolution solution-state NMR spectroscopy. (2008) Magn. Reson. Chem. 46(6), 508-517; Kim, I-L, Ralph, I., and Akiyama, T. Solution-state 2D NMR of Ball-milled Plant Cell Wall Gels in DMSO-d₆. (2008) BioEnergy Research 1(1), 56-66; Lu, F., and Ralph, J. Non-degradative dissolution and acetylation of ball-milled plant cell walls; high-resolution solution-state NMR. (2003) Plant J. 35(4), 535-544). The new transgenic plants can also be evaluated for a battery of functional agronomic characteristics such as lodging, yield, resistance to disease, resistance to insect pests, drought resistance, and/or herbicide resistance.

Determination of Stably Transformed Plant Tissues: To confirm the presence of the nucleic acids encoding terpene synthesizing enzymes in the regenerating plants, or seeds or progeny derived from the regenerated plant, a variety of assays may be performed. Such assays include, for example, molecular biological assays, such as Southern and Northern blotting and PCR; biochemical assays, such as detecting the presence of enzyme products, for example, by enzyme assays, by immunological assays (ELISAs and Western blots). Various plant parts can be assayed, such as trichomes, leaves, bracts, seeds or roots. In some cases, the phenotype of the whole regenerated plant can be analyzed.

Whereas DNA analysis techniques may be conducted using DNA isolated from any part of a plant, RNA may only be expressed in particular cells or tissue types and so RNA for analysis can be obtained from those tissues. PCR techniques may also be used for detection and quantification of RNA produced from introduced nucleic acids. PCR can also be used to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then this DNA can be amplified through the use of conventional PCR techniques. Further information about the nature of the RNA product may be obtained by Northern blotting. This technique will demonstrate the presence of an RNA species and give information about the integrity of that RNA. The presence or absence of an RNA species can also be determined using dot or slot blot Northern hybridizations. These techniques are modifications of Northern blotting and also demonstrate the presence or absence of an RNA species.

While Southern blotting may be used to detect the nucleic acid encoding the enzyme(s) in question, it may not provide information as to whether the preselected DNA segment is being expressed. Expression may be evaluated by specifically identifying the protein products of the introduced nucleic acids or evaluating the phenotypic changes brought about by their expression.

Assays for the production and identification of specific proteins may make use of physical-chemical, structural, functional, or other properties of the proteins. Unique physical-chemical or structural properties allow the proteins to be separated and identified by electrophoretic procedures, such as, native or denaturing gel electrophoresis or isoelectric focusing, or by chromatographic techniques such as ion exchange, liquid chromatography or gel exclusion chromatography. The unique structures of individual proteins offer opportunities for use of specific antibodies to detect their presence in formats such as an ELISA assay. Combinations of approaches may be employed with even greater specificity such as Western blotting in which antibodies are used to locate individual gene products that have been separated by electrophoretic techniques. Additional techniques may be employed to absolutely confirm the identity of the enzyme such as evaluation by amino acid sequencing following purification. Other procedures may be additionally used.

The expression of a gene product can also be determined by evaluating the phenotypic results of its expression. These assays also may take many forms including but not limited to analyzing changes in the chemical composition, morphology, or physiological properties of the plant. Chemical composition may be altered by expression of preselected DNA segments encoding storage proteins which change amino acid composition and may be detected by amino acid analysis.

Hosts

Terpenes, including diterpenes and terpenoids, can be made in a variety of host organisms either in vitro or in vivo. In some cases, the enzymes described herein can be made in host cells, and those enzymes can be extracted from the host cells for use in vitro. As used herein, a “host” means a cell, tissue or organism capable of replication. The host can have an expression cassette or expression vector that can include a nucleic acid segment encoding an enzyme that is involved in the biosynthesis of terpenes.

The term “host cell”, as used herein, refers to any prokaryotic or eukaryotic cell that can be transformed with an expression cassettes or vector carrying the nucleic acid segment encoding an enzyme that is involved in the biosynthesis of one or more terpenes. The host cells can, for example, be a plant, bacterial, insect, or yeast cell. Expression cassettes encoding biosynthetic enzymes can be incorporated or transferred into a host ceil to facilitate manufacture of the enzymes described herein or the terpene, diterpene, or terpenoid products of those enzymes. The host cells can be present in an organism. For example, the host cells can be present in a host such as a plant.

For example, the enzymes, terpenes, diterpenes, and terpenoids can be made in a variety of plants or plant cells. Although some of the enzymes described herein are from species of the mint family, the enzymes, terpenes, diterpenes, and terpenoids can be made in species other than in mint plants or mint plant cells. The terpenes, diterpenes, and terpenoids can, for example, be made and extracted from, whole plants, plant parts, plant ceils, or a combination thereof. Enzymes can conveniently, for example, be produced in bacterial, insect, plant, or fungal (e.g., yeast) ceils.

Examples of host cells, host tissues, host seeds and plants that may be used for producing terpenes and terpenoids (e.g., by incorporation of nucleic acids and expression systems described herein) include but are not limited to those useful for production of oils such as oilseeds, camelina, canola, castor bean, corn, flax, lupins, peanut, potatoes, safflower, soybean, sunflower, cottonseed, oil firewood trees, rapeseed, rutabaga, sorghum, walnut, and various nut species. Other types host cells, host tissues, host seeds and plants that can be used include fiber-containing plants, trees, flax, grains (maize, wheat, barley, oats, rice, sorghum, millet and rye), grasses (switchgrass, prairie grass, wheat grass, sudangrass, sorghum, straw-producing plants), softwood, hardwood and other woody plants (e.g., poplar, pine, and eucalyptus), oil (oilseeds, camelina, canola, castor bean, lupins, potatoes, soybean, sunflower, cottonseed, oil firewood trees, rapeseed, rutabaga, sorghum), starch plants (wheat, potatoes, lupins, sunflower and cottonseed), and forage plants (alfalfa, clover and fescue). In some embodiments the plant is a gymnosperm. Examples of plants useful tor pulp and paper production include most pine species such as loblolly pine, Jack pine. Southern pine, Radiata pine, spruce, Douglas fir and others. Hardwoods that can be modified as described herein include aspen, poplar, eucalyptus, and others. Plants useful for making biofuels and ethanol include corn, grasses (e.g., miscanthus, switchgrass, and the like), as well as trees such as poplar, aspen, pine, oak, maple, walnut, rubber tree, willow, and the like. Plants useful for generating forage include legumes such as alfalfa, as well as forage grasses such as bromegrass, and bluestem. In some cases, the plant is a Brassicaceae or other Solanaceae species. In some embodiments, the plant is not a species of Arabidopsis, for example, in some embodiments, the plant is not Arabidopsis thaliana.

Additional examples of hosts cells and host organisms include, without limitation, tobacco cells such as Nicotiana benthamiana, Nicotiana tabacum, Nicotiana rustica, Nicotiana excelsior, and Nicotiana excelsiana cells; cells of the genus Escherichia such as the species Escherichia coif cells of the genus Clostridium such as the species Clostridium ljungdahlii, Clostridium autoethanogenum or Clostridium kluyveri; cells of the genus Corynebacterium such as the species Corynebacterium glutamicum; cells of the genus Cupriavidus such as the species Cupriavidus necator or Cupriavidus metallidurans; cells of the genus Pseudomonas such as the species Pseudomonas fluorescens, Pseudomonas putida or Pseudomonas oleavorans; cells of the genus Delftia such as the species Delftia acidovorans; cells of the genus Bacillus such as the species Bacillus subtilis; cells of the genus Lactobacillus such as the species Lactobacillus delbrueckii; or cells of the genus Lactococcus such as the species Lactococcus lactis.

“Host cells” can further include, without limitation, those from yeast and other fungi, as well as, for example, insect ceils. Examples of suitable eukaryotic host cells include yeasts and fungi from the genus Aspergillus such as Aspergillus niger, from the genus Saccharomyces such as Saccharomyces cerevisiae; from the genus Candida such as C. tropicalis, C. albicans, C. cloacae, C. guillermondii, C. intermedia, C. maltosa, C. parapsilosis, and C. zeylenoides; from the genus Pichia (or Komagataella) such as Pichia pastoris; from the genus Yarrowia such as Yarrowia lipolytica: from the genus Issatchenkia such as Issathenkia orientalis; from the genus Debaryomyces such as Debaryomyces hansenii; from the genus Arxula such as Arxula adenoinivorans; or from the genus Kluyveromyces such as Khiyveromyces lactis or from the genera Exophiala, Mucor, Trichoderma, Cladosporium, Phanerochaete, Cladophialophora, Paecilomyces, Scedosporium, and Ophiostoma.

In some cases, the host cells can have organelles that facilitate manufacture or storage of the terpenes, diterpenes, and terpenoids. Such organelles can include lipid droplets, smooth endoplasmic reticulum, plastids, trichomes, vacuoles, vesicles, plastids, and cellular membranes. During and after production of the terpenes, diterpenes, and terpenoids these organelles can be isolated as a semi-pure source of the of the terpenes, diterpenes, and terpenoids.

The Diterpene Skeletons of Lamiaceae and how to Make them

Enzymes responsible for all new skeletons were not specifically located, but considering the known skeletons and diTPS activities, the inventors have deduced how diverse skeletons arise and what strategies may be used for finding the enzymes responsible. All of the six diterpene skeletons with a known biosynthetic route in Lamiaceae contain a decalin core: Sk2, and Sk4 (FIG. 1B-1C) are skeletons of the direct products of TPS-c enzymes, while Sk1, Sk3, Sk6, and Sk14 fire skeletons of the products a TPS-e enzyme acting on a labdadiene diphosphate (Sk4) precursor.

Many diterpene skeletons with an intact decalin core can be made by as-yet undiscovered diTPSs from the TPS-c and TPS-e subfamilies, for example through methyl shifts during cyclization. Examples of diTPSs that catalyze methyl shifts are foe TPS-c enzymes SdKPS and ArTPS2 which produce the clerodane skeleton (Sk2), and the TPS-e enzyme OmTPS5 which has a product with the abietane skeleton (Sk3). The same mechanisms may form skeletons such as Sk8 and Sk12. Other decalin-containing skeletons, for example the nor-diterpenes (missing one or more methyl side chains, e.g. Sk7) are can be made by oxidative decarboxylation occurring after the TPS steps. Ring rearrangements catalyzed by TPS-e enzymes also have precedent, for example foe generation of ent-kaurene (with skeleton Sk1) or cur-atiserene (with skeleton Sk14) from ent-CPP (with skeleton Sk4), but always preserve the decaline core structure.

Diterpenoids lacking a decalin core are taxonomically restricted within Lamiaceae, with no single skeleton being reported in more than two clades (FIG. 1B). Many can be explained as modifications occurring after the TPS steps to decalin-containing skeletons. Cytochrome P450 driven ring contraction, akin to that in the gibberellin pathway, can play a role in the formation of skeletons such as Sk13. Ring opening and ring expansion may also occur, for example in pathways to compounds with the 6,7-seco-kaurane (Sk5), and icetaxane (Sk9) skeletons, respectively. Skeletons such as cembrane (Sk11), lacking any apparent biosynthetic connection to a decalin core can arise from diTPSs outside the TPS-c and TPS-e subfamilies. In Euphorbiaceae and Solanaceae, where cembranoid compounds are common, the relevant TPSs come from the TPS-a subfamily. Elucidation of pathways to the remaining diterpene skeletons in Lamiaceae will depend on broadening the search to new genera and species and new TPS subfamilies, eventually moving beyond TPSs to look at cytochromes P450 and other enzyme families.

Implications for Biotechnology

Arrays of compounds can be produced by combining class IT diTPSs with different class I diTPSs. Particularly prolific enzymes for combinatorial biosynthesis have been Cyc2 from the bacterium Streptomyces griseolosporeus (Hamano et al. J Biol Chem 277(40):37098-37104 (2002); Dairi et 1. J Bacteriol 183(20):6085-6094 (2001)), which generates alkene moieties on prenyl-diphosphate substrates, and SsSS, which installs an alcohol at the 13 position and a double bond at the 14 position; both of these enzymes have demonstrated activity on 12 different class II enzyme products. The inventors have found that SsSS is also active on the products of PcTPS1 and ArTPS2. In addition, the inventors have found class 1 enzymes that provide routes to products that previously were biosynthetically inaccessible or poorly accessible. OmTPS3 is active on class II products with a labdane skeleton and normal absolute configuration, typically generating a trans-methyl-pentadiene moiety, as in 11, 34, and 24. An enzyme with similar activity, producing 24 and 34, was recently reported from the bacterium Streptomyces cyslabdanicus (Yarnada et al. The Journal of Antibiotics 69(7):515-523 (2016); Ikeda et al. J Ind Microbiol Biotechnol 43(2-3):325-342 (2016)) but was not tested against additional substrates. LITPS4 produces sandaracopimaradiene [27] from 31, with greater specificity than the earlier enzyme, Euphorbia peplus TPS8 (Andersen-Ranberg et al. Angew Chem Int Ed 55(6):2142-2146 (2016)). Finally, OmTPS5 enables efficient and specific production of palustradiene [29] from 31. The other known biosynthetic route to 29 is as a minor spontaneous degradation product of 13-hydroxy-8(14)-abietane from Picea abies levopimaradiene/abietadiene synthase and related enzymes.

ArTPS2 is of particular interest for applications in agricultural biotechnology. Neo-clerodane diterpenoids, particularly those with an epoxide moiety at the 4(18)-position, have garnered significant attention for their ability to deter insect herbivores. The 4(18)-desaturated product of ArTPS2 could be used in biosynthetic or semisynthetic routes to potent insect antifeedants.

Definitions

As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Also, as used herein, “and/or” refers to, and encompasses, any and all possible combinations of one or more of the associated listed items. Unless otherwise defined, all terms, including technical and scientific terms used in the description, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

The term “about”, as used herein, can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range.

The term “enzyme” or “enzymes”, as used herein, refers to a protein catalyst capable of catalyzing a reaction. Herein, the term does not mean only an isolated enzyme, but also includes a host cell expressing that enzyme. Accordingly, the conversion of A to B by enzyme C should also be construed to encompass the conversion of A to B by a host cell expressing enzyme C.

The term, “heterologous” when used in reference to a nucleic acid refers to a nucleic acid that has been manipulated in some way. For example, a heterologous nucleic acid includes a nucleic acid from one species introduced into another species. A heterologous nucleic acid also includes a nucleic acid native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to a non-native promoter or enhancer sequence, etc.). Heterologous nucleic acids can include cDNA forms of a nucleic acid; the cDNA may be expressed in either a sense (to produce mRNA) or anti-sense orientation (to produce an anti-sense RNA transcript that is complementary to the mRNA transcript). For example, heterologous nucleic acids can be distinguished from endogenous plant nucleic acids in that the heterologous nucleic acids are typically joined to nucleic acids comprising regulatory elements such as promoters that are not found naturally associated with the natural gene for the protein encoded by the heterologous gene. Heterologous nucleic acids can also be distinguished from endogenous plant nucleic acids in that the heterologous nucleic acids are in an unnatural chromosomal location or are associated with portions of the chromosome not found in nature (e.g., the heterologous nucleic acids are expressed in tissues where the gene is not normally expressed).

The terms “identical” or percent “identity”, as used herein, in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 75% identity, 80% identity, 85% identity, 90% identity, 95% identity, 97% identity, 98% identity, 99% identity, or 100% identity in pairwise comparison). Sequence identity can be determined by comparison and/or alignment of sequences for maximum correspondence over a comparison window, or over a designated region as measured using a sequence comparison algorithm, or by manual alignment and visual inspection. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity. A “reference sequence” is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence.

As used herein, a “native” nucleic acid or polypeptide means a DNA, RNA or amino acid sequence or segment that has not been manipulated in vitro, i.e., has not been isolated, purified, amplified and/or modified.

As used herein, the term “plant” is used in its broadest sense. It includes, but is not limited to, any species of grass (fodder, ornamental or decorative), crop or cereal, fodder or forage, fruit or vegetable, fruit plant or vegetable plant, herb plant, woody plant, flower plant or tree. It is not meant to limit a plant to any particular structure. It also refers to a unicellular plant (e.g. microalga) and a plurality of plant cells that are largely differentiated into a colony (e.g. volvox) or a structure that is present at any stage of a plant's development. Such structures include, but are not limited to, a seed, a tiller, a sprig, a stolen, a plug, a rhizome, a shoot, a stem., a leaf, a flower petal, a fruit, et cetera.

The term “plant tissue” includes differentiated and undifferentiated tissues of plants including those present in roots, shoots, leaves, pollen, seeds and tumors, as well as cells in culture (e.g., single cells, protoplasts, embryos, callus, etc.). Plant tissue may be in planta, in organ culture, tissue culture, or cell culture.

As used herein, the term “plant part” as used herein refers to a plant structure or a plant tissue, for example, pollen, an ovule, a tissue, a pod, a seed, a leaf and a cell. Plant parts may comprise one or more of a tiller, plug, rhizome, sprig, stolen, meristem, crown, and the like. In some instances, the plant part can include vegetative tissues of the plant.

The terms “in operable combination,” “in operable order,” and “operably linked” refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a coding region (e.g., gene) and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.

As used herein the term “terpene” includes any type of terpene or terpenoid, including for example any monoterpene, diterpene, sesquiterpene, sesterterpene, triterpene, tetraterpene, polyterpene, and any mixture thereof.

The term “transgenic” when used in reference to a plant or leaf or vegetative tissue or seed for example a “transgenic plant,” transgenic leaf,” “transgenic vegetative tissue,” “transgenic seed,” or a “transgenic host cell” refers to a plant or leaf or tissue or seed that contains at least one heterologous or foreign gene in one or more of its cells. The term “transgenic plant material” refers broadly to a plant, a plant structure, a plant tissue, a plant seed or a plant cell that contains at least one heterologous gene in one or more of its cells.

As used herein, the term “wild-type” when made in reference to a gene refers to a functional gene common throughout an outbred population. As used herein, the term “wild-type” when made in reference to a gene product refers to a functional gene product common throughout an outbred population. A functional wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the “normal” or “wild-type” form of the gene.

The following non-limiting Examples describe some procedures that can be performed to facilitate making and using the invention.

Example 1: Materials and Methods

This Example illustrates some of the materials and methods used in the development of the invention.

Data Mining

A subset of the NAPRALERT database including all the occurrences of diterpenoids in mint species was obtained. NAPRALERT reports chemical mimes, but not structures. For Lamiaceae, the species reported in NAPRALERT largely overlap with those from the Dictionary of Natural Products (DNP), which does include structures. A simplifying assumption was therefore made that each unique name represents a unique compound, and structures for the 3080 Lamiaceae diterpenes in NAPRALERT were not ail located due to the deficiencies of the NAPRALERT database.

For SISTEMAT, structure files were obtained by redrawing the structures from the publication by Alvarenga et al. (2001) into MarvinSketch (ChemAxon, Budapest, Hungary). The occurrence counts were obtained by transcribing the association table into a spreadsheet. A publicly available digital version of SISTEMAT, called SISTAMATX exists (see website at sistematx.ufpb.br/), but there is no option for bulk downloads, limiting assessment of its completeness or the ability to cross-reference it with other data. For the present work, the proprietary DNP therefore appeared to be one of the only viable option for many analyses.

Lamiaceae diterpene structures were obtained from the DNP by searching for them through the DNP web interface. Additional compounds were found by searching for individual species names for which transcriptome data was available. This additional search step was used because some species have been reclassified between families, or their family is not correctly annotated in the DNP. Records for all the Lamiaceae diterpenes were downloaded and converted into a spreadsheet using a Python script. Species names were extracted from the Biological Source field in a semi-automated method. The DNP contains structural information in the form of TUPAC International Chemical Identifier (InChI) strings (Heller et al. J Cheminform 7 (2015)). In most cases, the DNP InChIs do not include stereochemical information, so for consistency, all stereochemical information was ignored. Skeletons were extracted from, the structures using the RDKit (see website at rdkit.org) Python interface. Briefly, all bonds were converted into single bonds, bonds involving at least one non-carbon atom were broken, and the fragment with a carbon-count closest to 35 was retained as the skeleton. The resulting skeletons were then manually examined to correct those where the algorithm chose the wrong fragment, for example, a small number of diterpenoids are attached to acyl chains of more than 20 carbons, in which case the algorithm would incorrectly select the acyl chain as the skeleton; the diterpenoid was therefore selected instead. There are a few cases where sesquiterpenes or other terpenes seemed to have been misannotated in DNP as diterpenes, and those sesquiterpenes or other terpenes were left in the dataset, but their presence or absence does not significantly change any of the analyses.

For all three databases, genus and species names were cross-referenced to TaxIDs from the NCBI Taxonomy database (Federhen Nucleic Acids Res 40(D1): D136-D143 (2012)), first by automated text comparisons, then by manual inspection of un-matched names. Genus level TaxID assignments were possible for every entry in NAPRALERT and the DNP, but in some cases, species-level TaxID assignments were not possible, so species-level analyses were avoided.

Phylogenetic Trees

Peptide sequences were aligned using Clustal Omega (v. 1.2.1) (Sievers et at., Molecular Systems Biology 7:539 (2011)) and maximum likelihood trees were generated using RAxML (v. 8.2.11) (Stamatakis Bioinformatics 30(9): 1312-1313 (2014)) using automatic model selection and 1000 bootstrap iterations. Tree visualizations were generated using ETE3 (Huerta-Cepas Mol Biol Evol 33(6):1635-1638 (2016)).

Plant Material, RNA Isolation and cDNA Synthesis

The following types of plants were obtained from different commercial nurseries or botanical gardens: Ajuga reptans L., Hyptis suaveolens (L.) Poit., Leonotis leonurus (L.) R.Br., Mentha splcata L., Nepeta mussinii Spreng. ex Henckel, Origanum majorana L., Perovskia atriplicifolia Benth., Plectranthus barbatus, Pogostemon cablin (Blanco) Benth., Prunella vulgaris L., and Salvia officinalis L. The plants were grown in a greenhouse under ambient photoperiod and 24° C. day/17° C. night temperatures. Nicotiana benthamiana were grown in a greenhouse under 16 h light (24° C.) and 8 h dark (17° C.) regime.

Total RNA from leaf tissues of A. reptans, N. mussini, L. leonurus, P. atriplicifolia, and S. officinalis was extracted using methods described by Hamberger et al. (Plant Physiology 157(4): 1677-1695 (2011)). Total RNA from leaves of P. vulgaris, M. spicata, P. cablin, H. sauveolans, O. majorana was extracted using the Spectrum Plant Total RNA Kit (Sigma-Aldrich, St. Louis, Mo., USA). RNA extraction was followed by DNase I digestion using DNA-Free™ DNA Removal Kit (Thermo Fisher Scientific, Waltham, Mass., USA). First-strand cDNAs were synthesized from 5 μg of total RNA, with oligo(dT) primer, using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, Mass., USA). cDNA was diluted 5-fold and used as template for cloning of full length cDNAs. See Table 2 for primers and other oligonucleotides.

Characterization of diTPS Genes by Transient Expression in N. benthamiana

Full length coding sequences of diTPSs were cloned into pEAQ-HT vector (Sainsbury et al., 2009; kindly provided by Prof. G. Lomonossoff, John Innes Centre, UK) using In-Fusion® HD Cloning Plus (Takara Bio, California, USA). pEAQ-HT vector contains a copy of anti-post transcriptional gene silencing protein p19 that suppresses the silencing of transgenes (Voinnet et al. The Plant Journal 33(5):949-956). Expression vectors carrying full length coding sequence of candidate diTPS genes were transformed into the LBA4404 A. tumefaciens strain by electroporation. DXS and GGPPS are known to be the rate limiting enzymes in GGPP biosynthesis and have been shown to substantially increase the production of diterpenes in N. benthamiana system. Therefore, the Plectranthus barbatus 1-deoxy-D-xylulose 5-phosphate synthase (CfDXS) (genhank accession: KP889115) and geranylgeranyl diphosphate synthase (CfGGPPS) (genhank accession: KP889114) coding regions were cloned, and a chimeric polyprotein was created with a LP4-2A hybrid Sinker peptide between CfDXS and CfGGPPS. LP4/2A contains the first nine amino acids of LP4 (a linker peptide originating from a natural polyprotein occurring in seeds of Impatiens balsamina) and 20 amino acids of the self-processing FMDV 2A (2A is a peptide from the foot-and-mouth disease virus).

The transformed A. tumefaciens were subsequently transferred to 1 mL SOC media and grown for 1 hour at 28° C. 100 μL cultures were transferred to LB-agar solid media containing 50.0 μg/mL rifampicin and 50.0 μg/mL kanamycin and grown for 2 days. A single colony PCR positive clone was transferred to 10 mL LB media in a falcon tube containing 50.0 μg/mL rifampicin and 50.0 μg/mL kanamycin and grown at 28° C. over-night (at 225 rpm). About 1% of the primary culture was transferred to 25 mL of fresh LB media and grown overnight. Cells were pelleted by centrifugation at 4000×g for 15 min and resuspended in 10 mL water containing 200 μM acetosyringone. Cells were diluted with water-acetosyringone solution to a final OD₆₀₀ of 1.0 and incubated at 28° C. for 2-3 hours to increase the infectivity. Equal volumes of culture containing the plasmids with cDNA encoding different diTPS genes were mixed. Each combination of A. tumefaciens culture mixture was infiltrated into independent 4-5 weeks old N. benthamiana plants. Plants were grown for 5-7 days in the greenhouse before metabolite extraction. Leaf discs of 2 cm diameter (approximately 0.1 g fresh weight) were cut from the infiltrated leaves. Diterpenes were extracted in 1 mL n-hexane with 1 mg/L 1-eicosene as internal standard (IS) at room temperature overnight in an orbital shaker at 200 rpm. Plant material was collected by centrifugation and the organic phase transferred to GC vials for analysis.

In-Vitro Enzyme Activity Assays

To confirm the biosynthetic products obtained in N. benthamiana, diTPS combinations were tested in in vitro assays as described by Pateraki et al. (Plant Physiol 164(3): 1222-1236 (2014)). TargetP (Emanuelsson et al. Journal of Molecular Biology 300(4):1005-1016 (2000)) was used for prediction of the plastidial target sequence. Pseudo mature variants versions of HsTPS1, ArTPS2, PcTPS1, OmTPS3, OmTPS5, SsSS, CfTPS1, CfTPS2 and codon optimized GTPS3 (IDT, USA), lacking the predicted plastidial targeting sequences were cloned in pET-28b(+) (EMD Millipore, Burlington, Mass.), then expressed and purified from E. coli. The pET_diTPS constructs were transformed into chemically competent OverExpress™ C41(DE3) cells (Lucigen, Middleton, Wis., USA), the cells were inoculated in a starter culture with terrific broth medium and 50 μg mL⁻¹ kanamycin, then grown overnight. About 1% of the starter culture was used to inoculate 50 mL terrific broth medium having 50 μg mL⁻¹ kanamycin, and the culture was grown at 37° C. with mixing at 200 rpm until the OD₆₀₀ reached 0.4. Cultures were grown at 16° C. until an OD₆₀₀ of approximately 0.6-0.8 was achieved at which point cultures were induced by 0.2 mM IPTG. Expression was allowed to proceed overnight, and cells were harvested by centrifugation at 5000 g/4° C. for 15 minutes. Cell pellets were resuspended in lysis buffer containing 20 mM HEPES, pH 7.5, 0.5 M NaCl, 25 mM Imidazole, 5% [v/v] glycerol, using one protease inhibitor cocktail tablet per 100 mL (Sigma Aldrich, St. Louis, Mo., USA). Lysozyme (0.1 mg per liter) was added to the cell pellet, and the mixture was gently shaken for 30 min, then lysed by sonication. Cell lysate was centrifuged for 25 min at 14000 g, and the supernatant was subsequently used for purification of the recombinant proteins. Proteins were purified on 1-mL His SpinTrap columns (GE Healthcare Life Sciences, Piscataway, N.J., USA) using elution buffer (HEPES, pH 7.5, 0.5 M NaCl, 5% [v/v] glycerol, 350 mM Imidazole and 5 mM dithiothreitol [DTP]) and desalted on PD MiniTrap G-25 columns (GE Healthcare, Life Sciences, Piscataway, N.J., USA) with a desalting buffer (20 mM HEPES, pH 7.2, 350 mM NaCl, 5 mM DTT, 1 mM MgCl₂, 5% [v/v] glycerol). In-vitro diTPS assays were performed by adding 15 μM GGPP and 50-100 μg purified enzymes in 400 μL enzyme assay buffer (50 mM HEPES, pH 7.2, 7.5 mM MgCl₂, 5% [v/v] glycerol, 5 mM DTT). 500 mL n-hexane (Fluka GC-MS grade) containing 1 ng/ml 1-eicosene as internal standard was gently added as an overlay onto the reaction mix. Assays were incubated for 60-120 min at 30° C. with mixing at approximately 75 rpm, and the hexane overlay was subsequently removed by centrifugation at 1500×g for 15 min before proceeding for GC-MS analysis.

Metabolite Analysis of O. majorana

Fresh leaf, stem, root, and flowers (20 to 50 mg) of O. majorana were harvested. Flowers were further separated with forceps into two parts, the green part (“calyx”), and the rest of the flower (“corolla”). Tissues were extracted overnight in 500 μL of methyl tert-butyl ether. Extracts were concentrated to 100 μL and subjected to GC-MS analysis.

Compound Purification

For bulk production of diterpenes for structural determination, 15-30 N. benthamiana plants were vacuum infiltrated with diTPS combinations as well as CfGGPPS and CfDXS (46). After 5 days, 100-200 g (fresh weight) of leaves were subjected to two rounds of overnight extractions in 500 mL hexane, which was then concentrated using a rotary evaporator. Compounds were purified on silica gel columns using a mobile phase of hexane with 0-20% ethyl-acetate, in some cases, additional rounds of column purification, or preparative TLC using a hexane/ethyl-acetate or chloroform/methanol mobile phase, were necessary to obtain compounds of sufficient purity for structural determination by NMR.

GC-MS

All GC-MS analyses were performed on an Agilent 7890A GC with an Agilent VF-5 ms column (30 m×250 μm×0.25 μm, with 10 m EZ-Guard) and an Agilent 5975C detector. For N. benthamiana and in-vitro assays, the inlet was set to 250° C. splitless injection, using helium carrier gas with column flow of 1 mL/min. The oven program was 45° C. hold 1 min, 40° C./min to 230° C. 7° C./min to 320° C., hold 3 min. The detector was activated after a four-minute solvent delay. For analysis of O. majorana extracts, conditions were the same, except that the solvent cutoff was set to six minutes to allow monoterpenes to pass, and the oven program was a 45° C. hold for 1 min., 40° C./min to 200° C. 5° C./min to 260° C. 40° C./min to 320° C., with a hold for 3 min.

NMR and Optical Rotation

The NMR spectra for trans-biformene (Yamada et al. The Journal of Antibiotics 69(7):515-523 (2016)) were measured on a Bruker AVANCE 900 MHz spectrometer. Ail other spectra were measured on an Agilent DirectDrive2 500 MHz spectrometer. AH NMR was done in CDCl₃ solvent. The CDCl₃ peaks were referenced to 7.24 ppm and 77.23 ppm for ¹H and ¹³C spectra, respectively. To aid in the interpretation of NMR spectra, the NAPROC-13 (Lopez-Perez et al. Bioinformatics 23(23):3256-3257 (2007)), and Spektraris (Fischedick et ah, Phytochemistry 113:87-95 (2015)) databases were used. Reconstruction of ¹³C spectra from the literature was performed with MestReNova (Mestrelab Research, Santiago de Compostela, Spain). Optical rotation was measured in chloroform at ambient temperature using a Perkin Elmer Polarimeter 341 instrument.

TABLE 2 List of synthetic oligonucleotides Primer Name (gene) Sequence Amplification of full length genes from   cDNA synthesized from plant tissues total RNA ZmAN2-F ATGGTTCTTTCATCGTCTTGCACA (ZmAN2) (SEQ ID NO:61) ZmAN2-R TTATTTTGCGGCGGAAACAGGTTCA (ZmAN2) (SEQ ID NO: 62) CfTPS2-F AGATTGAGGATTCCATTGAGTACGTGAAGG (CfTPS2) (SEQ ID NO: 63) CfTPS2-R GAAGTTTAATATCCTTCATTCTTTATTACA (CfTPS2) (SEQ ID NO:64) CfTPS3-F AGCTCCATTCAACTAGAGTCATGTCGT (CfTPS3) (SEQ ID NO:65) CfTPS3-R TTCATCTGGCTTAACTAGTTGCTGACAC (CfTPS3) (SEQ ID NO:66) CfTPS16-F TTAAAGTACTCTCTCAAAGAGTACTTTGG (CfTPS16) (SEQ ID NO:67) CfTPS16-R GCGACCAACCATCATACGACT (CfTPS16) (SEQ ID NO:68) LITPS1-F AATGGCCTCCACTGCATCCACTCTA (LITPS1) (SEQ ID NO:69) LITPS1-R CCATACTCATTCAACTGGTTCGAACA (LITPS1) (SEQ ID NO:70) LITPS4-F AGCCTGTGTACTCGAAATGTC (LITPS4) (SEQ ID NO:71) LITPS4-R CAAGAGGATGATTCATGTACCAAC (LITPS4) (SEQ ID NO:72) SoTPS1-F TCTCTTTCAAGAATATCCCCTCTC (SoTPS1) (SEQ ID NO:73) SoTPS1-R GGCATTCAATGATTTTGAGTCG (SoTPS1) (SEQ ID NO:74) ArTPS1-F AAATGGCCTCTTTGTCCACTCTC (ArTPS1) (SEQ ID NO:75) ArTPS1-R TTACGCAACTGGTTCGAAAAGCA (ArTPS1) (SEQ ID NO:76) ArTPS2-F TAATGTCATTTGCTTCCCAAGCCA (ArTPS2) (SEQ ID NO:77) ArTPS2-R GGCCTAGACTATACCTTCTCAAACAA (ArTPS2) (SEQ ID NO:78) ArTPS3-F AATGTCACTCTCGTTCACCATCAA (ArTPS3) (SEQ ID NO:79) ArTPS3-R ACTTCAAGAGGATGAAGTGTTTAGG (ArTPS3) (SEQ ID NO:80) PaTPS1-F CTCCAAAACTCGGGCCGGTAAAT (PaTPS1) (SEQ ID NO:81) PaTPS1-R TACGTATTTCCTCACAATCGAGCA (PaTPSI) (SEQ ID NO:82) PaTPS3-F CTAGAAATGTTACTTGCGTTCAAC (PaTPS3) (SEQ ID NO:83) PaTPS3-R GGGTAAGAGTTGAATTTAGATGTCT (PaTPS3) (SEQ ID NO:84) NmTPS1-F ATGACTTCAATATCCTCTCTAAATTTGAGC (NmTPS1) (SEQ ID N0:85) NmTPS1-R GAATATAGTAATCAGACGACCGGTCCA (NmTPS1) (SEQ ID N0:86) NmTPS2-F GCCATATCATGTCTCTTCCGCTCT (NmTPS2) (SEQ ID NO:87) NmTPS2-R TTATTCATGCACCTTAAAATCCTTGAGAG (NmTPS2) (SEQ ID NO:88) OmTPS1-F ATGACCGATGTATCCTCTCTTCGT (OmTPS1) (SEQ ID N0:89) OmTPS1-R AAACACTCACATAACCGGCCCAA (OmTPS1) (SEQ ID NO:90) OmTPS3-F GTCCTTGCTTTCGGAATACT (OmTPS3) (SEQ ID N0:91) OmTPS3-R GAAGTGATCTACAAGGATTCATAAA (OmTPS3) (SEQ ID NO:92) OmTPS4-F TCATTGATTTGCCCTGCATCCAC (OmTPS4) (SEQ ID N0:93) OmTPS4-R CAAAGCTAGTGCTGCTTCTGATT (0mTPS4) (SEQ ID N0:94) OmTPS5-F ATGGTATCTGCATGTCTAAAACTCAA (0mTPS5) (SEQ ID NO:95) OmTPS5-R CTTTCTCTCTCTTGTGCATCTTAGT (OmTPS5) (SEQ ID NO:96) MsTPS1-F ACGTTCATCTTCAATGAGTTCCA (MsTPS1) (SEQ ID N0:97) MsTPS1-R TACGTGTATGTCGATCTGTTCCAAT (MsTPS1) (SEQ ID NO:98) PcTPS1-F CATGTCATTTGCTTCTCAATCAC (PcTPS1) (SEQ ID NO:99) PcTPS1-R CCCATTATCTAAAAGTCTACATCACC (PcTPS1) (SEQ ID NO:100) HsTPS1-F TCCTCATAAAGCAATGGCGTATA (HsTPS1) (SEQ ID NO:101) HsTPS1-R CTAAGATTCAGACAATGGGCTCA (HsTPS1) (SEQ ID NO:102) EpTPS8-F GCAGACGCCAATCTTTCTTGGT (EpTPS8) (SEQ ID NO:103) EpTPS8-R TTATGAAGTTAAAAGGAGTGGTTCGTTGAC (EpTPS8) (SEQ ID N0:104) PVTPS1-F GGAACGAGAAATGTCACTCAC (PVTPS1) (SEQ ID NO:105) PVTPS1-R TTCTAGTTTCTCACAGAAGTCAA (PVTPS1) (SEQ ID NO:106) LP4-2A Ver.1 TCAAATGCAGCAGACGAAGTTGCTACT sequence CAACTTTTGAATTTTGACTTGCTGAAGTT GGCTGGTGATGTTGAGTCAAACCCTGGACCT (SEQ ID NO:107) Cloning of full length diTPS genes into pEAQ-HT  for transient expression in N. benthamiana pEAQ_Infusion TTCTGCCCAAATTCGATGGGGTCTCTATC _CfTPS1-F CACTATGA (CfTPS1) (SEQ ID NO:108) pEAQ_Infustion AGTTAAAGGCCTCGATCAGGCGACTGGTTCG _CfTPS1-R AA (CfTPS1) AAGTA (SEQ ID NO:109) pEAQ_Infusion TTCTGCCCAAATTCGATGTCGCTCGCCTT _SsSCS-F CAAC (SsSS) (SEQ ID NO:110) pEAQ_Infusion  AGTTAAAGGCCTCGATCAAAAGACAAAGGAT _SsSCS-R T (SsSS) TCATA (SEQ ID NO:111) pEAQ_Infusion TTCTGCCCAAATTCGATGGTTCTTTCATCG _ZrnAN2-F TCTT (ZmAN2) GCAC (SEQ ID No:l12) pEAQ_Infusion AGTTAAAGGCCTCGATTATTTTGCGGCGGAA _ZmAN2-R AC (ZmAN2) AGGT (SEQ ID NO:113) pEAQ_Infusion TTCTGCCCAAATTCGATGAAAATGTTGATG _CfTPS2-F ATCA (CfTPS2) AAAGT (SEQ ID NO:114) pEAQ_Infusion_CfTP AGTTAAAGGCCTCGATCAGACCACTGGTT S2-R CAAA (CfTPS2) TAGTA (SEQ ID NO:115) pEAQ_Infusion_CfTP TTCTGCCCAAATTCGATGTCGTCCCTCGCC S3-F GGC (CfTPS3) AACCT (SEQ ID NO:116) pEAQ_Infusion AGTTAAAGGCCTCGACTAGTTGCTGACACAA _CfTPS3-R CT (CfTPS3) CATT (SEQ ID NO:117) pEAQ_Infusion TTCTGCCCAAATTCGATGCAGGCTTCTATGTC _CfTPS16-F ATCT (CfTPS16) (SEQ ID NO:118) pEAQ_infusion AGTTAAAGGCCTCGATCATACGACTGGTTCA _CfTPS16-R AA (CfTPS16) CATT (SEQ ID NO:119) pEAQ_Infusion TTCTGCCCAAATTCGATGGCCTCCACTGCATC _LITPS1-F C (LITPS1) (SEQ ID NO:120) pEAQ_Infusion AGTTAAAGGCCTCGATCATTCAACTGGTTCGA _LITPS1-R ACAA (LITPS1) (SEQ ID NO:121) pEAQ_Infusion TTCTGCCCAAATTCGATGATTCCTAATCCCGA _LITPS2-F AA (LITPS2) (SEQ ID NO:122) pEAQ_Infusion AGTTAAAGGCCTCGATTACATTGGCAATCCG _LITPS2-R ATGAA (LITPS2) (SEQ ID NO:123) pEAQ_Infusion TTCTGCCCAAATTCGATGTCGGTGGCGTTCAA _LITPS4-F CCT (LITPS4) (SEQ ID NO:124) pEAQ_Infusion AGTTAAAGGCCTCGATCAAGAGGATGATTCA _LITPS4-R TG (LITPS4) TACC (SEQ ID NO:125) pEAQ_Infusion TTCTGCCCAAATTCGATGTCCCTCGCCTTCAA _SoTPS1-F CG (SoTPS1) (SEQ ID NO:126) pEAQ_/Infusion AGTTAAAGGCCTCGATCATTTGCCACTCACAT _SoTPS1-R TT (SoTPS1) (SEQ ID NO:127) pEAQ_infusion TTCTGCCCAAATTCGATGGCCTCTTTGTCCAC _ArTPS1-F TTTCC (ArTPS1) (SEQ ID NO:128) pEAQ_/Infusion AGTTAAAGGCCTCGATCACGCAACTGGTTCG _ArTPS1-R AAA (ArTPS1) AGA (SEQ ID NO:129) pEAQ_Infusion TTCTGCCCAAATTCGATGTCATTTGCTTCCCA _ArTPS2-F AG (ArTPS2) CCAC (SEQ ID NO:130) pEAQ_Infusion AGTTAAAGGCCTCGACTAGACTACCTTCTCAA _ArTPS2-R ACA (ArTPS2) ATAC (SEQ ID NO:131) pEAQ_Infusion TTCTGCCCAAATTCGATGTCACTCTCGTTCAC _ArTPS3-F CATCA (ArTPS3) (SEQ ID NO:132) pEAQ_Infusion AGTTAAAGGCCTCGATCAAGAGGATGAAGTG _ArTPS -R TTTAG (ArTPS3) (SEQ ID NO:133) pEAQ_Infusion TTCTGCCCAAATTCGATGACCTCTATGTCCTC _PaTPS1-F TCTAA (PaTPS1) (SEQ ID NO:134) pEAQ_Infusion AGTTAAAGGCCTCGATCATACGACCGGTCCA _PaTPS1-R AAC (PaTPS1) AGT (SEQ ID NO:135) pEAQ_Infusion TTCTGCCCAAATTCGATGTTACTTGCGTTCAA _PaTPS3-F CATA (PaTPS3) AGC (SEQ ID NO:136) pEAQ_Infusion AGTTAAAGGCCTCGATTAATTAGGTAGGTAG _PaTPS3-R AGGG (PaTPS3) GTT (SEQ ID NO:137) pEAQ_Infusion ATATTCTGCCCAAATTCGATGACTTCAATATC __NmTPS1-F CTCT (NmTPS1) CTAAATTTGAGCAATG (SEQ ID NO:138) pEAQ_Infusion CAGAGTTAAAGGCCTCGATCAGACGACCGGT _NmTPS1-R CCAA (NmTPS1) (SEQ ID NO:139) pEAQ_Infusion TTCTGCCCAAATTCGATGTCTCTTCCGCTCTC _NmTPS2-F CTCT (NmTPS2) (SEQ ID NO:140) pEAQ_Infusion GATAAGTTAAAGGCCTCGATTATTCATGCACC _NmTPS2-R TTA (NmTPS2) AAATCCTTGAGAGC (SEQ ID NO:141) pEAQ_Infusion TTCTGCCCAAATTCGATGACCGATGTATCCTC _OmTPS1-F TCTTC (OmTPS1) (SEQ ID NO:142) pEAQ_Infusion AGTTAAAGGCCTCGATCACATAACCGGCCCA _OmTPS1-R AACA (OmTPS1) (SEQ ID NO:143) pEAQ_Infusion TTCTGCCCAAATTCGATGGCGTCGCTCGCGTT _OmTPS3-F CAC (SEQ ID NO:144) (OmTPS3) pEAQ_Infusion AGTTAAAGGCCTCGACTACAAGGATTCATAA _OmTPS3-R ATT (OmTPS3) AAGGA (SEQ ID NO:145) pEAQ_Infusion TTCTGCCCAAATTCGCGAATGTCACTCGCCTT _OmTPS4-F CAGC (OmTPS4) (SEQ ID NO:146) pEAQ_Infusion AGTTAAAGGCCTCGAGCTAGGAGCTTAGGGT _OmTPS4-R TT (0mTPS4) TCAT (SEQ ID NO:147) pEAQ_Infusion TTCTGCCCAAATTCGATGGTATCTGCATGTCT _OmTPS5-F AAA (0mTPS5) (SEQ ID NO:148) pEAQ_Infusion AGTTAAAGGCCTCGATCATGAAGGAATTGAA _OmTPS5-R GGAA (OmTPS5) (SEQ ID NO:149) pEAQ_Infusion TTCTGCCCAAATTCGATGAGTTCCATTCGAAA _MsTPS1-F TTT (MsTPS1) AAGT (SEQ ID NO:150) pEAQ_Infusion AGTTAAAGGCCTCGATCACTTGAGAGGCTCA _MsTPS1-R AAC (MsTPS1) ATCAT (SEQ ID NO:151) pEAQ_Infusion TTCTGCCCAAATTCGATGTCATTTGCTTCTCA _PcTPS1-F AT (PCTPS1) CAC (SEQ ID NO:152) pEAQ_Infusion AGTTAAAGGCCTCGACTACATCACCCTCTCAA _PcTPS1-R ACA (PcTPS1) ATAC (SEQ ID NO:153) pEAQ_Infusion TTCTGCCCAAATTCGATGGCGTATATGATATC _HsTPS1-F TAT (HsTPS1) TTCAAATCTC (SEQ ID NO:154) pEAQ_/Infusion AGTTAAAGGCCTCGATCAGACAATGGGCTCA _HsTPS1-R AAT (HsTPS1) AGAAC (SEQ ID NO:155) pEAQ_Infusion TTCTGCCCAAATTCGATGCAAGTCTCTCTCTC _EpTPS8-F C (EpTPS8) CTCA (SEQ ID NO:156) pEAQ_Infusion AGTTAAAGGCCTCGATTATGAAGTTAAAAGG __EpTPS8-R AG (EpTPS8) TGGTT (SEQ ID NO:157) pEAQ_Infusion TTCTGCCCAAATTCGCGAATGTCACTCACTTT _PVTPS1-F CA (PVTPS1) ACG (SEQ ID NO:158) pEAQ_Infusion AGTTAAAGGCCTCGAGCTAGTTTCTCACAGA _PVTPS1-R AG (PVTPS1) TCAA (SEQ ID NO:159) Cloning of diTPS genes into pET-28 b  (+30) for E. coli expression AGGAGATATACCATGGCCGAGATTCGAGTG pET28_CfTPS1-F CCAC (CfTPS1) (SEQ ID NO:160) GGTGGTGGTGCTCGAAGGCGACTGGTTCGAA pET28_CfTPS1-R AAG (CfTPS1) TAC (SEQ ID NO:161) AGGAGATATACCATGGATTTCATGGCGAAAA pET28__SsSS-F TGAA (SsSS) AGAGA (SEQ ID NO:162) GGTGGTGGTGCTCGAAAAAGACANAGGATTT pET28__SsSS-R CATAT (SsSS) (SEQ ID NO:163) AGGAGATATACCATGCAAATTCGTGGAAAGC pET28__CfTPS2-F AAAG (cfTPS2) ATCAC (SEQ ID NO:164) GGTGGTGGTGCTCGAAGACCACTGGTTCAAA pET28_CfTPS2-R TAG (CfTPS2) AACT (SEQ ID NO:165) AGGAGATATACCATGTCTAAATCATCTGCAG pET28_CfTPS3-F CTGT (CfTPS3) (SEQ ID NO:166) GGTGGTGGTGCTCGAAGTTGCTGACACAACT pET28__CfTPS3-R CATT (CfTPS3) (SEQ D NO:167) pET28_OmTPS3-F AGGAGATATACCATGACCGTCAAATGCTAC (OmTPS3) (SEQ ID NO:168) GGTGGTGGTGCTCGAACAAGGATTCATAAAT pET28_OmTPS3-R TAAG (OmTPS3) (SEQ ID NO:169) pET28_OmTPS5-F AGGAGATATACCATGACTGTCAAGTGCAGC (OmTPS5) (SEQ ID NO:170) pET28_OmTPS5-R GGTGGTGGTGCTCGAATGAAGGAATTGAAG (OmTPS5) (SEQ ID NO:171) AGGAGATATACCATGTTTATGCCCACTTCCAT pET28_PcTPS1-F TAA (pcTPS1) ATGTA (SEQ ID NO:172) GGTGGTGGTGCTCGAACATCACCCTCTCAAA pET28__PcTPS1-R CAA (PcTPS1) TACTTTGG (SEQ ID NO:173) AGGAGATATACCATGGTAGCAAAAGTGATCG pET28_HsTPS1-F AGAG (HsTPS1) CCGAGTTA (SEQ ID NO:174) GGTGGTGGTGCTCGAAGACAATGGGCTCAAA pET28_HsTPS1-R TAGA (HsTPS1) ACTTTAAAT (SEQ. ID NO:175)

Example 2: Diversity of Diterpenoids in Lamiaceae

To help determine the most promising species in which to find previously unknown hut useful diterpene synthase (diTPS) activities, a dataset of diterpene occurrences in Lamiaceae species and a dataset of functionally characterized diTPS genes from Lamiaceae were generated. Information about diterpene occurrence was collected from three sources, SISTEMAT, DNP, and NAPRALERT. SISTEMAT (Vestri et al. Phytochemistry 56(6):583-595 (2001)) contains Lamiaceae diterpenes reported up to 1997, including 91 unique carbon skeletons (the core alkanes, disregarding all desaturation, acyl-side chains, heteroatoms, and stereochemistry) from. 295 species and 51 genera. An electronic copy of SISTEMAT was not available, so it was reconstructed based on the figures and tables in the paper.

The Dictionary of Natural Products (DNP; see website at dnp.chemnetbase.com, accessed Jan. 11, 2018) includes a wealth of information on diterpenes from Lamiaceae, including full structures and the species where those structures have been reported. NAPRALERT (Loub et al., J Chem Inf Comput Sci 25(2):99-103 (1985)) identifies compounds by their common name rather than their structure or skeleton, but it does associate the compounds to genus and species names, and gives various other information, such as the tissue where the compound was found.

To enable comparison among the databases, and cross-referencing with transcriptome and enzyme data, all genus and species manes were converted into TaxIDs from the NCBI Taxonomy database (Federhen Nucleic Acids Res 40(D1): D136-D143 (2012)). To put structure occurrences into clearer evolutionary context, each genus was annotated as a member of one of the 12 monophyletic clades that form the backbone of Lamiaceae, as delineated by Li and colleagues (Li et al. Scientific Reports 6:34343 (2016)).

In the context of diTPSs, examination of skeletons can be helpful because the skeleton often resembles the diterpene synthase product more obviously than a highly decorated downstream product would. Therefore, the skeletons were extracted from the DNP structures. An example of such skeleton extraction is shown below, where Table 3A provides an example of which class I diTPS generate which products when using a N. benthamiana transient expression. Bold numbers refer to assigned compound numbers; “np” indicates that the combination was tested but no product was detected; indicates that the combination was not tested. The following are newly identified enzymes: LITPS1, HsPS1, PcTPS1, ArTPS2, OmTPS1, ArTPS3, LITPS4, MsTPS1, NmTPS2, OmTPS3, OmTPS4, OmTPS5, PaTPS3, PvTPS1, and SoTPS1.

TABLE 3A Index of Enzyme Types and Products Observed in Transient Expression Assays CfTPS1 CfTPS2 LlTPS1 ZmAN2 HsPS1 PcTPS1 ArTPS2 OmTPS1 Enzyme [31] [10] [5] [16] [21] [25] [38] [31] ArTPS3 32 8 1, 2, 3 np — — np — LlTPS4 27 8 1, 2, 3 np — — — — MsTPS1 27 8 3 np — — np — NmTPS2 np np np 19 — — np — OmTPS3 34 11  1, 2 np 24 — np 34 OmTPS4 33 8 1, 2, 3, 4 20 — — — 33 OmTPS5 29 8 1, 2, 3 np — — np 29 PaTPS3 32 8 1, 2, 3 np — — — — PvTPS1 32 8 1, 2, 3 np — — — — SoTPS1 32 8 1, 2, 3 np — — — — CfTPS3 32 8 1, 2, 3 np 22 np np 32 SsSS 33 — 4 20 23 26 37 —

Table 3B provides an example of an index of new class II diTPS enzymes and the products identified by functional assays of these enzymes using the N. benthamiana transient expression assay. The products were identified by GC-MS chromatography of hexane extracts from. N. benthamiana transient expression assays that expressed new (+)-CPP synthases or new class II diTPSs along with reference combinations.

TABLE 3B Products Identified for New Class II diTPS Enzymes Enzyme Product ArTPS1 Copalyl-PP [31] CfTPS16 Copalyl-PP [31] NmTPS1 Copalyl-PP [31] OmTPS1 Copalyl-PP [31] PaTPS1 Copalyl-PP [31] ArTPS2 Neo-cleroda-4(18), 13E-dienyl-PP [38] HsTPS1 Labda-7,13E-dienyl-PP [21] LlTPS1 Peregrinol-PP [7] PcTPS1 Ent-labda-8,13E-dienyl-PP [25]

Using data like that obtained in Tables 3A and 3B, a labdane skeleton was extracted from the forskolin structure shown below by deleting all heteroatoms, desaturations, and stereochemistry.

A tabulation of the skeletons from. SISTEMAT and DNP was therefore generated.

The three databases were relatively consistent in their estimations of the diversity and distribution of diterpenes and diterpene skeletons, as illustrated in Table 4 and FIG. 1B, 1D.

TABLE 4 Comparison of different sources for data about Lamiaceae diterpene chemotaxonomy DNP NAPRALERT SISTEMAT Genera 67 60 44 Species 342 378 — Diterpene 3336 3080 — names Diterpene 3268 — — structures Diterpene 229 — 91 skeletons

A total of 239 skeletons are represented, with five, the kaurane (Sk1), clerodane (Sk2), abietane (Sk3), labdane (Sk4), and pimarane (Sk6) being, by far, the most widely distributed and accounting for most of the total structures (Table 4, FIG. 1B-1C). The clerodane skeleton, for example, has the widest distribution, having been reported in 27 genera representing 9 of the 12 backbone clades, absent only in Tectona and two clades from which no diterpenes have yet been reported. The large number of less common, taxonomically restricted skeletons, including over 100 skeletons with only one associated compound (FIG. 1C), indicted to the inventors that searching across many species and genera would be a good strategy for finding diterpene synthases with new activities.

Example 3: Identifying Candidate Diterpene Synthase Genes

Through a comprehensive literature search, a reference set was built of known Lamiaceae diTPSs and their activities. Fifty-four functional diTPSs have been reported in this family, which correspond to thirty class II and 24 class I enzymes. Combinations of these diterpene synthases account for twenty-seven distinct products represented by six different skeletons, the five widely distributed skeletons, Sk1-4 and Sk6, as well as the less common atisane (Sk14) skeleton. This leaves 233 skeletons for which the biosynthetic route remains unknown. Further, a single skeleton can correspond to multiple distinct diTPS products, so there is also a possibility of finding new diTPS activities for skeletons already accounted for by known enzymes.

BLAST homology searches (Camacho et al. BMC Bioinformatics 10:421 (2009)) were performed to the list of Lamiaceae diTPSs to mine 48 leaf transcriptomes made available by the Mint Genome Project (Boachon et al. Molecular Plant. (2018)) for candidate diTPSs. The number of diTPS candidates was cross-referenced to the number of diterpenes and diterpene skeletons reported from each species and genus (Table 5). Table 5 shows species from which diTPSs were selected for cloning, the total number of diTPS candidate sequences, and the number of unique diterpene structures and skeletons for those species, based on DNP.

TABLE 5 Species from which diTPSs were Isolated diTPS Full name Code hits Diterpenes Skeletons Ajuga reptans Ar 5 13 2 Hyptis suaveolens Hs 7 4 1 Leonotis leonurus Ll 5 14 2 Mentha spicata Ms 5 0 0 Nepeta mussinii Nm 3 0 0 Origanum majorana Om 5 0 0 Perovskia Pa 5 2 2 atriplicifolia Plectranthus Cf 5 50 10 barbatus Pogostemon cablin Pc 2 0 0 Pruneila vulgaris Pv 1 1 1 Salvia officinalis So 5 13 5

A phylogenetic tree was generated from the peptide sequences from the reference set, alongside those from the new transcriptome data, including established substrates and products for each enzyme (FIG. 3A, 3B-1 to 3B-4). Candidate genes were selected from species such as Mentha x spicata and Origanum majorana, where the transcriptome data showed multiple candidate diTPSs likely existed but where few or no diterpene product structures have been reported. Genes were also selected that had relatively low homology to known enzymes. In this way, the inventors attempted to evenly cover of the sequence homology space. A few candidates from Piectranthus and Salvia were also selected based on the great diversity of diterpenes that have been reported from these genera.

Example 4: Characterization of Class II diTPSs

FIG. 3A presents a summary of Lamiaceae diTPS structures and activities reported from previous work, together with the newly characterized diTPS activities identified as described herein. Class II activities were established based on tire activities of extracts from Nicotiana benthamiana that transiently expressed the new genes, compared with the activities of known diTPS (or combinations) that were similarly expressed.

Class II diTPS products retained the diphosphate group from the GGPP substrate. When expressed in-vivo, whether in E. call or N. benthamiana, without a compatible class I diTPS, a diphosphate product degrades to the corresponding alcohol, presumably by the action of non-specific endogenous phosphatases. Due to difficulties in purifying and structurally characterizing diphosphate class II products it is customary in the field to instead characterize the alcohol derivatives (Heskes et al. Plant J 93(5):943-958 (2018); Pelot et al. Plant J 89(5):885-897 (2017)), which is the approach taken in this study. For clarity, the alcohol has been indicated by appending an “a” to the compound number, for example, 16a refers to ent-copalol.

ArTPS1, PaTPS1, NmTPS1, OmTPS1, and CfTPS1 were identified as (+)-copalyl diphosphate ((+)-CPP) [31] synthases by comparison to products of Plectranthus barbatus (synonym Coleus forskohli) CfTPS1, and the reference combination of CfTPS1 combined with CfTPS3, yielding miltiradiene (Pateraki et al. Plant Physiol 164(3):1222-1236 (2014)). LITPS1 was identified as a peregrinol diphosphate (PgPP) [5] synthase based on a comparison of products with Marrubium vulgare MvCPS1 (Zerbe et al. Plant J 79(6):914-927 (2014)), and MvCPS1 combined with M. vulgare 9,13-epoxylabdene synthase (MvELS), and Salvia sclarea sclareol synthase (SsSS) (Jia et al. Metabolic Engineering 37:24-34 (2016)).

Table 6 illustrates the distribution among selected Lamiaceae clades of diterpenes with various structural patterns. Blue enzyme names are placed according to the pattern they install and the clade of the species they were cloned from. A solid line indicates that only compounds with the bond-type shown at that position are counted. A dashed line indicates that all types of bonds and substituents are counted at that position. Based on data from the DNP.

TABLE 6: Table 6A: Lamiaceae clades of diterpenes with various structural patterns. Clerodane Cleroda-4(18)-ene 4(18)-epoxy-Clerodane

Ajugoideae 317 (ArTPS2) 6 206 Lamioideae  32  3 1 Nepetoideae 132  1 1 Scutellarioideae 160 19 78 Viticoideae  1  0 0 All clades 668 31 289 Table 6B: Lamiaceae clades of diterpenes with various structural patterns. Clerodane-3-ene Labdane

Ajugoideae  23  3 Lamioideae  25 201 Nepetoideae  84  60 Seutellarioideae  44  0 Viticoideae   0  37 All clades 189 300 Table 6C: Lamiaceae clades of diterpenes with various structural patterns. Labda-8-ene Labda-7-ene

Ajugoideae  2 0 Lamioideae (PcTPS1)27 5 Nepetoideae  1 (HsTPS1) 1 Scutellarioideae  0 0 Viticoideae  2 2 AU clades 33 9

HsTPS1 was identified as a (55, 95, 105) labda-7,13E-dienyl diphosphate [21] synthase based on comparison to the product of an enzyme from Grindelia robusta, GrTPS2 (Zerbe et al. The Plant Journal 83(5):783-793 (2015)), and by NMR of the alcohol derivative [21a]. Normal absolute stereochemistry was assigned to the HsTPS1 product based on the optical rotation of 21a, [α]_(D)+8.3° (c. 0.0007, CHCl₃) (c.f. lit. [α]_(D)+5°, c. 1.0, CHCl₃ (Urones et al. Phytochemistry 35(3):713-719 (1994)); [α]_(D) ²⁵+12°, c. 0.69, CHCl₃ (Suzuki et al. Phytochemistry 22(5): 1294-1295 (1983)). When HsTPS1 was expressed in N. benthamiana, labda-7,13(16), 14-triene [22] was formed, which seemed to be enhanced by co-expression with CfTPS3. The combination of HsTPS1 with OmTPS3 produced labda-7,12E, 14-triene [24] (Roengsumran et al. Phytochemistry 50(3):449-453 (1999)), which has previously been accessible only by combinations of bacterial enzymes (Yamada et al. The journal of Antibiotics 69(7):515-523 (2016)). Labdanes with a double bond at the 7-position have not been reported in H. suaveolens, and such labdanes do not seem to be common in Lamiaceae. Of nine compounds with the labdane skeleton and a double bond at position-7 (Table 6) only one was from the same clade as H. suaveolens. (13E)-ent-labda-7,13-dien-15-oic acid, from Isodon scoparius (Xiang et al. Helvetica Chimica Acta 87(11):2860-2865 (2004)), has the opposite absolute stereochemistry to the HsTPS1 product, likely not deriving from aparalog of HsTPS1 because absolute stereochemistry of a skeleton is not known to change after the diTPS steps.

ArTPS2 was identified as a (5R,8R,9S,10R) neo-cleroda-4(18),13E-dienyl diphosphate [38] synthase. The combination of ArTPS2 and SsSS generated neo-cleroda-4(18),14-dien-13-ol [37] (FIG. 4A). The structures of compounds 37 and 38a were determined by NMR. The analysis included a comparison of compound 37 to chelodane (Rudi et al. J Nat Prod 55(10): 1408-1414 (1992)), which based on small differences in ¹³C shifts, may be a stereoisomer of compound 37 at the 13 position, and a comparison of the NMR results for compound 38a with the NMR of its enantiomer (Ohaski et al. Bioorganic & Medicinal Chemistry Letters 4(24):2889-2892 (1994)). There were 20 to 19, and 20 to 17 NOE interactions in the NMR spectra of 37 and 38a, which closely resembled those reported for (−)-kolavelol [36a] (Pelot et al. Plant J 89(5):885-897 (2017)), indicating that the stereochemistry may be 5R,8R,9S,10R. The “neo” absolute configuration was established through optical rotation of 38a, [α]_(D)+30° (c. 0.0025, CHCl₃) (c.f. lit. [α]_(D)+20.9°, c. 0.7, CHCl₃) (Monaco et al. Rendiconto della Academia delle scienze fisiche e matematiche 48:465-470 (1982)).

Previously reported clerodane diTPSs from Lamiaceae produce kolavenyl diphosphate [36] (Heskes et al. Plant J 93(51:943-958 (2018); Chen et al. 1 Exp Bot 68(5):1109-1122 (2017); Pelot et al. Plant J 89(5):885-897 (2017)), and kolavenyl diphosphate [36] has a double bond at the 3-position. Clerodanes with desaturation at position-3 are spread throughout multiple clades but are most common in Nepetoideae (Table 6A-6C), which includes Salvia divinorum. Clerodanes with a double bond at the 4(18)-position are rare by comparison, but those with a 4(18)-epoxy moiety, make up nearly half of the clerodanes reported in Lamiaceae, including two-thirds of those reported from the Ajugoideae clade (Table 6A-6C), one of which is clerodin (Barton et al. J Chem Soc:5061-5073 (1961)) and from which the clerodane skeleton gets its name. Neo-cleroda-4(18),13E-dienyl diphosphate is a logical biosynthetic precursor for the 4(18)-epoxy clerodanes. It is unclear if any of the previously described diTPSs directly produce an epoxide moiety.

PcTPS1 was identified as a (10R)-labda-8,13E-dienyl diphosphate [25] synthase. The structure was established by comparison of ¹³C NMR of compound 25a to previously reported spectra (Suzuki et al. Phytochemistry 22(5): 1294-1295 (1983)). The 10R (ent-) absolute stereochemistry was established by optical rotation of compound 25a [α]_(D)−64° (c. 0.0008, CHCl₃), (c.f. lit. [α]_(D) ²⁵−71.2°, c. 1.11, CHCl₃) (Arima et al. Tetrahedron: Asymmetry 18(14): 1701-1711 (2007)). The combination of PcTPS1 and SsSS, both in-vitro, and in N. benthamiana expression produced (10R)-labda-8,14-en-13-ol [26] (FIG. 4B), the structure of which was determined by comparison of ¹³C NMR to a published spectrum (Wu & Lin Phytochemistry 44(1):101-105 (1997)). The double bond between positions 8 and 9 is present in 33 distinct compounds isolated from Lamiaceae (Table 6A-6C), most of which occur in the Lamioideae clade, which includes Pogostemon cablin, the source of PcTPS1. Absolute stereochemistries of the reported compounds are mixed, with some in the normal configuration (Boalino et al. J Nat Prod 67(4):714-717 (2004)), and others in the cut-configuration (Gray et al. Phytochemistry 63(4):409-413 (2003)). As normal configuration 9-hydroxy labdanes tire also abundant in Lamioideae, it is possible that the normal configuration 8(9) desaturated labdanes arise from dehydratase activities downstream of a PgPP synthase (MvCPS1 and its paralogs), while those in the cur-configuration arise from paralogs of PcTPS1. Another possibility is that some of the 8(9) desaturated labdanes reported as having normal absolute stereochemistry are actually ent-labdanes that were mis-assigned, as has occurred in at least one documented case (Gray et al. Phytochemistry 63(4):409-413 (2003)).

Example 5: Characterization of Class I diTPSs

Class I diTPS candidates were characterized by transient expression in N. benthamiana in combination with four class II enzymes:

-   -   CfTPS1, a (+)-CPP [31] synthase;     -   CfTPS2, a labda-13-en-8-ol diphosphate ((+)-8-LPP) [10] synthase         (Pateraki et al. Plant Physiol 164(3): 1222-1236 (2014);     -   LITPS1, a PgPP [5] synthase; or     -   Zea mays ZmAN2, an ent-copalyl diphosphate (ent-CPP) [16]         synthase (Harris et al. Plant Mol Biol 59(6):881-894 (2005)).         Substrates accepted by each enzyme and the products are         indicated in FIG. 2B and FIG. 5. NmTPS2 was identified as an         ent-kaurene [19] synthase, converting ent-CPP into ent-kaurene         (identified using Physcomitrella patens extract as a standard         (Zhan et al. Plant Physiology and Biochemistry 96:110-114         (2015))), but not showing activity with any other substrate. The         only other enzyme to show activity with ent-CPP was OmTPS4,         which produced ent-manool [20], just as SsSS produces from         ent-CPP.

PaTPS3, PvTPS1, SoTPS1, ArTPS3, OmTPS4, LITPS4, OmTPS5, and MsTPS1 converted (+)-8-LPP to 13R-(+)-manoyl oxide [8], verified by comparison to the product of CfTPS2 and CfTPS3 (Pateraki et al. Plant Physiol 164(3): 1222-1236 (2014)). OmTPS3 produced trans-abienol [11]. The trans-abienol structure was determined by NMR, with the stereochemistry of the 12(13)-double bond supported by comparison of the NOESY spectrum to that of a commercial standard for cis-abienol (Toronto Research Chemicals, Toronto Canada). The trans-abienol showed clear NOE correlation between positions 16 and 11, while the cis-abienol standard showed correlations between 14 and 11.

PaTPS3, PvTPS1, SoTPS1, and ArTPS3, LITPS4, and OmTPS5 converted PgPP to a combination of 1, 2, and 3, with some variation in the ratios between the products. Because perigrinol [5a] spontaneously degrades into 1, 2, and 3 under GC conditions (Zerbe et al. Plant J 79(6):914-927 (2014)), it was difficult to distinguish whether these enzymes have low activity, but specific products, or moderate activity with a mix of products. Nevertheless, differences in relative amounts of the products observed between LITPS1 alone and in combination with these class 1 enzymes suggest that they do have some activity on PgPP. OmTPS4 produced 1, 2, 3, turd 4. MsTPS1 produced only 3, and OmTPS3 produced only 1, and 2. PgPP products were established by comparison to MvCPS1, MvCPS1 with MvELS (Zerbe et al. Plant J 79(6):914-927 (2014)), and MvCPS1 with SsSS (Jia et al. Metabolic Engineering 37:24-34 (2016)).

PaTPS3, PvTPS1, SoTPS1, and ArTPS3 converted (+)-CPP to miltiradiene [32], similarly to CfTPS3. OmTPS4 produced manool [33], as compared to SsSS. LITPS4 and MsTPS1 produced sadaracopimaradiene [27], by comparison to a product from Euphorbia peplus EpTPS8 (Andersen-Ranberg et al. Angew Chem Int Ed 55(6):2142-2146 (2016)). OmTPS5 produced palustradiene [29], as compared to a minor product from Abies grandis abietadiene synthase (Vogel et al. J Biol Chem 271(38):23262-23268 (1996)). OmTPS3 produced trans-biformene [34], as established by comparison of ¹³C-NMR of compounds described by Bohlmann & Czerson, Phytochemistry 18(1): 115-118 (1979)), with a trans configuration further supported by clear NOE correlations between 16 and 11, and the absence of NOE correlations between 14 and 11.

Example 6: Origanum majorana Enzymes can Make Palustradiene and Other Diterpenoids

The class I enzymes from Origanum majorana, OmTPS3, OmTPS4, and OmTPS5 all produced different products from (+)-CPP, which itself is the product of OmTPS1 from the same species. Despite the apparent richness of activities of enzymes from O. majorana, no reports of diterpenes were located from that species either in database searches, or in a subsequent literature search.

To determine whether diterpene synthases are active in O. majorana, the products of enzyme combinations with extracts from O. majorana leaf, stem, calyx, corolla, and root were evaluated. Palustradiene [29], the product of OmTPS1 and OmTPS5, was detected in all tissues except roots (FIG. 6). In addition, two diterpene alcohols were detected in the stem., leaf, and calyx. One diterpene alcohol, could not be identified, but the other was a close match to palustrinol, the 19-hydroxy derivative of palustradiene, in the NTST17 spectral library. The structures of the palustrinol, and the 19-hydroxy derivative of palustradiene are shown below.

Example 7: Chiococca alba Enzymes can Make 13(R)-Epi-Dolabradiene and Other Compounds

This Example illustrates that enzymes from Chiococca alba can produce products such as ent-kaurene, ent-dolabradiene (13-epi-dolabradiene), and (13R)-ent-manoyl oxide.

Enzyme assays were prepared as described herein that separately or in combination contained the following enzymes and substrates:

-   -   class I terpene synthase enzyme from Chiococca alba (CaTPS1)         with SoTPS2, SbTPS1, and SbTPS2 and the substrate ent-copalyl         diphosphate.     -   class II terpene synthase enzyme from Chiococca alba (CaTPS2)         with substrate ent-labda-13-en-8-ol diphosphate     -   class III and class IV terpene synthase enzymes from Chiococca         alba (CaTPS3 and CaTPS4) with substrate ent-kaurene     -   class V terpene synthase enzyme from Chiococca alba (CaTPS5)         with substrate ent-dolabradiene     -   class I (−)-kolavenyl diphosphate synthase enzyme from Salvia         hispanica (ShTPS1) with substrate (−)-kolavenyl diphosphate     -   class I cleroda-4(18),13E-dienyl diphosphate synthase enzyme         from Teucrium canadense (TcTPS1) with substrate clerodadienyl         diphosphate     -   class I sclareol synthase enzyme from Salvia sclarea (SsSCS)         with substrate neo-clerodadienol.

FIG. 7 illustrates the activities of the newly obtained Chiococca alba terpene synthases CaTPS1-5. FIGS. 7A-7C show GC-MS-total ion and extracted ion chromatograms from in vivo assays within N. benthamiana that transiently expressed various combinations of enzymes. Mass spectra are shown below the chromatograms of FIG. 7A-7C for peaks (1) to (3) containing the following products of the enzymatic conversion: (1) ent-kaurene; (2) ent-dolabradiene (13-epi-dolabradiene); (3) (13R)-ent-manoyl oxide. The ent-dolabradiene was identified through extensive structural studies with NMR and the stereochemistry at C-13 was unequivocally corroborated by optical rotation. The ent-kaurene and (13R)-ent-manoyl oxide were identified through direct comparison with biosynthesized authentic standards with reference enzymes.

Compounds ent-dolabradiene (13-epi-dolabradiene) and (13R)-ent-manoyl oxide are plausible intermediates in the biosynthetic routes to the structurally unusual merilactone and ribenone, that have demonstrated activity against Leishmanina and potential anti-cancer activity (Piozzi, F., Bruno, M. Diterpenoids from Roots and Aerial Parts of the Genus Stachys Rec, Nat. Prod. 5, 1-11, (2011)).

Both merilactone and ribenone are detected in the root extract of C. alba.

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All patents and publications referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced patent or publication is hereby specifically incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such cited patents or publications.

The following statements are intended to describe and summarize various features of the invention according to the foregoing description provided in the specification and figures.

Statements:

-   -   1. An expression system comprising at least one expression         cassette having a heterologous promoter operably linked to a         nucleic acid segment encoding an enzyme with at least 90%         sequence identity to SEQ ID NOT, 3, 5, 7, 9, 11, 13, 15, 17, 19,         21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,         53, 57, 59, or 176     -   2. The expression system of statement 1, wherein at least one         expression cassette is within at least one expression vector.     -   3. The expression system of statement 1 or 2, wherein the         expression system comprises two, or three, or four, or five         expression cassettes or expression vectors, each expression         cassette encoding a separate enzyme.     -   4. The expression system of statement 1, 2 or 3, wherein the         expression system further comprises one or more expression         cassettes having a promoter operably linked to a nucleic acid         segment encoding an enzyme that can synthesize isopentenyl         diphosphate (IPP), dimethylallyl diphosphate (DMAPP), or         geranylgeranyl diphosphate (GGPP).     -   5. The expression system of statement 1-3 or 4, wherein the         expression system has at least one expression cassette having a         constitutive promoter.     -   6. The expression system of statement 1-3 or 4, wherein the         expression system has at least one expression cassette having an         inducible promoter.     -   7. The expression system of statement 1-5 or 6, wherein the         expression system has at least one expression cassette having a         CaMV 35S promoter, CaMV 19S promoter, nos promoter, AclhI         promoter, sucrose synthase promoter, α-tubulin promoter,         uhiquitin promoter, actin promoter, cab promoter, PEPCase         promoter, R gene complex promoter, CYP71D16 trichome-specific         promoter, CBTS (cembratrienol synthase) promotor, Z10 promoter         from a 10 kD zein protein gene, Z27 promoter from a 27 kD zein         protein gene, plastid rRNA-operon (rrn) promoter, tight         inducible pea rbcS gene, RUBISCO-SSU light-inducible promoter         (SSU) from tobacco, or rice actin promoter.     -   8. A host cell comprising the expression system of statement 1-6         or 7, which is heterologous to the host cell.     -   9. The host cell of statement 8, which is a plant cell, an algae         cell, a fungal cell, a bacterial cell, or an insect cell.     -   10. The host cell of statement 8 or 9, which is a Nicotiana         benthamiana, Nicotiana tabacum, Nicotiana rustica, Nicotiana         excelsior, Nicotiana excelsiana, Escherichia coli, Clostridium         ljungdahlii, Clostridium autoethanogemum, Clostridium kluyveri,         Corynebacterium glutamicum, Cupriavidus necator, Cupriavidus         metallidurans; Pseudomonas fluorescens, Pseudomonas putida,         Pseudomonas oleavorans; Delftia acidovorans, Bacillus subtilis,         Lactobacillus delbrueckii, Lactococcus lactis, Aspergillus         niger, Saccharomyces cerevisiae, Candida tropiccdis, Candida         albicans, Candida cloacae, Candida guillermondii, Candida         Intermedia, Candida maltosa, Candida parapsilosis, Candida         zeylenoides, Pichia pastoris, Yarrowia lipolytica, Issathenkia         orientalis, Debaryomyces hansenii, Arxula adenoinivorans,         Kluyveromyces lactis, or Exophiala, Mucor, Trichoderma,         Cladosporium, Phanerochaete, Cladophialophora, Paecilomyces,         Scedosporium, or Ophiostoma cell.     -   11. The host cell of statement 8, 9 or 10, which is a Nicotiana         benthamiana.     -   12. A method of synthesizing a terpene comprising incubating a         host cell that has the expression system of any of statements         1-7.     -   13. A method for synthesizing a terpene comprising incubating a         host cell comprising a heterologous expression system that         includes at least one expression cassette having a heterologous         promoter operably linked to a nucleic acid segment encoding an         enzyme with at least 90% sequence identity to SEQ ID NO:1, 3, 5,         7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,         39, 41, 43, 45, 47, 49, 51, 53, 57, 59, or 176.     -   14. A method for synthesizing a terpene comprising incubating a         terpene precursor with an enzyme with at least 90% sequence         identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,         23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,         57, 59, or 176.     -   15. The method of statement 12, 13 or 14, wherein the terpene is         a compound of formula I, II, or lit:

-   -   wherein         -   each R₁ can separately be hydrogen or lower alkyl;         -   R₂ can be hydrogen, lower alkyl, hydroxy, a bond to an             adjacent ring carbon, or form a C₄-C₆ cycloheteroalkyl with             R₃;         -   R₃ can be a branched C₅-C₆ alkyl with 0-2 double bonds, can             form a C₄-C₆ cycloheteroalkyl with R₂; can form a cycloalkyl             with R₄, or can form a cycloheteroalkyl ring with R₄,             wherein the C₅-C₆ alkyl can optionally have one hydroxy,             phosphate or diphosphate substituent, and wherein each             cycloalkyl or cycloheteroalkyl ring can have 0-2 double             bonds, and each cycloalkyl or cycloheteroalkyl ring can have             0-2 alkyl or 0-2 alkene substituents;         -   R₄ can be hydrogen, lower alkyl, lower alkene, hydroxy, a             carbon bonded to R₉, an oxygen bonded to R₉, form a             cycloalkyl ring with R₃, or form a cycloheteroalkyl ring             with R₃, wherein each cycloalkyl ring or cycloheteroalkyl             ring can have 0-2 double bonds, and each cycloalkyl ring or             cycloheteroalkyl ring can have 0-2 alkyl or 0-2 alkene             substituents;         -   R₅ can be hydrogen, hydroxy, lower alkyl, a lower alkene, a             bond with an adjacent carbon, form a cycloalkyl ring with a             ring atom of a ring formed by R₃ and R₄, wherein the             cycloalkyl ring can have 0-2 double bonds, and the             cycloalkyl ring can have 0-2 alkyl or 0-2 alkene             substituents;         -   each R₆ can separately be hydrogen, lower alkyl, lower             alkene, or form a bond with an adjacent carbon;         -   R₇ can be lower alkyl, lower alkene, or form a cycloalkyl             ring with a R₅,         -   R₈ can be lower alkyl, hydroxy, phosphate, diphosphate, or             form a bond with an adjacent carbon; and         -   R₉ can be hydrogen, lower alkyl, lower alkene, ═CH₂,             hydroxy, phosphate, diphosphate, form a bond with an             adjacent carbon, form a cycloalkyl ring with R₄, or form a             cycloheteroalkyl ring with R₄, wherein each cycloalkyl ring             or cycloheteroalkyl ring can have 0-2 double bonds, and each             cycloalkyl ring or cycloheteroalkyl ring can have 0-2 alkyl             or 0-2 alkene substituents.     -   16. The method of statement 12-14 or 15 wherein the terpene is a         compound

or a combination thereof.

-   -   17. The method of statement 12-15 or 16, wherein the terpene is         any of the following compounds:

-   -   wherein:

-   -   18. The method of statement 12-16 or 17, wherein the terpene is         at least one of the following compounds:

-   -   or         -   wherein:

-   -   19. The method of statement 12-17 or 18 wherein the terpene         precursor is geranylgeranyl diphosphate (GGPP).     -   20. A compound selected from:

-   -   -   wherein:

-   -   21. A reaction mixture comprising one or more of the following:

-   -   wherein:

The specific methods, devices and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and the methods and processes are not necessarily restricted to the orders of steps indicated herein or in the claims.

Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.

The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims and statements of the invention.

The invention has been described broadly and genetically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. 

1. An expression system comprising at least one expression cassette having a heterologous promoter operably linked to a nucleic acid segment encoding an enzyme with at least 95% sequence identity to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 57, 59, or
 176. 2. The expression system of claim 1, wherein the expression system comprises at least two, or three, or four, or five expression cassettes or expression vectors, each expression cassette encoding a separate enzyme.
 3. The expression system of claim 1, wherein the expression system further comprises one or more expression cassettes having a promoter operably linked to a nucleic acid segment encoding an enzyme that can synthesize isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP), or geranylgeranyl diphosphate (GGPP).
 4. The expression system of claim 1, wherein the expression system has at least one expression cassette having a constitutive or and inducible promoter.
 5. The expression system of claim 1, wherein the expression system has at least one expression cassette having a CaMV 35S promoter, CaMV 19S promoter, nos promoter, AdhI promoter, sucrose synthase promoter, α-tubulin promoter, ubiquitin promoter, actin promoter, cab promoter, PEPCase promoter, R gene complex promoter, CYP71D16 trichome-specific promoter, CBTS (cembratrienol synthase) promotor, Z10 promoter from a 10 kD zein protein gene, Z27 promoter from a 27 kD zein protein gene, plastid rRNA-operon (rrn) promoter, light inducible pea rbcS gene, RUBISCO-SSU light-inducible promoter (SSU) from tobacco, or rice actin promoter.
 6. A host cell comprising the expression system of claim 1, which is heterologous to the host cell.
 7. The host cell of claim 6, which is a plant cell, an algae cell, a fungal cell, a bacterial cell, or an insect cell.
 8. The host cell of claim 6, which is a Nicotiana benthamiana, Nicotiana tabacum, Nicotiana rustica, Nicotiana excelsior, Nicotiana excelsiana, Escherichia coli, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium kluyveri, Corynebacterium glutamicum, Cupriavidus necator, Cupriavidus metallidurans; Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas oleavorans; Delftia acidovorans, Bacillus subtilis, Lactobacillus delbrueckii, Lactococcus lactis, Aspergillus niger, Saccharomyces cerevisiae, Candida tropicalis, Candida albicans, Candida cloacae, Candida guillermondii, Candida intermedia, Candida maltosa, Candida parapsilosis, Candida zeylenoides, Pichia pastoris, Yarrowia lipolytica, Issathenkia orientalis, Debaryomyces hansenii, Arxula adenoinivorans, Kluyveromyces lactis, or Exophiala, Mucor, Trichoderma, Cladosporium, Phanerochaete, Cladophialophora, Paecilomyces, Scedosporium, or Ophiostoma cell.
 9. A plant or seed comprising the host cell of claim
 6. 10. A method for synthesizing a terpene comprising incubating a terpene precursor with an enzyme with at least 90% sequence identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 57, 59, or
 176. 11. The method of claim 10, wherein the precursor is isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP), geranylgeranyl diphosphate (GGPP), or a combination thereof.
 12. The method of claim 10, which comprises incubating a host cell that expresses comprising a heterologous expression system comprising at least one expression cassette having a heterologous promoter operably linked to a nucleic acid segment encoding an enzyme with at least 90% sequence identity to SEQ ID NO: SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 57, 59, or
 176. 13. The method of claim 10 or 12, wherein the terpene is a compound of formula I, II, or III:

wherein each R₁ can separately be hydrogen or lower alkyl; R₂ can be hydrogen, lower alkyl, hydroxy, a bond to an adjacent ring carbon, or form a C₄-C₆ cycloheteroalkyl with R₃; R₃ can be a branched C₅-C₆ alkyl with 0-2 double bonds, can form a C₄-C₆ cycloheteroalkyl with R₂; can form a cycloalkyl with R₄, or can form a cycloheteroalkyl ring with R₄, wherein the C₅-C₆ alkyl can optionally have one hydroxy, phosphate or diphosphate substituent, and wherein each cycloalkyl or cycloheteroalkyl ring can have 0-2 double bonds, and each cycloalkyl or cycloheteroalkyl ring can have 0-2 alkyl or 0-2 alkene substituents; R₄ can be hydrogen, lower alkyl, lower alkene, hydroxy, a carbon bonded to Ry, an oxygen bonded to R₉, form a cycloalkyl ring with R₃, or form a cycloheteroalkyl ring with R₃, wherein each cycloalkyl ring or cycloheteroalkyl ring can have 0-2 double bonds, and each cycloalkyl ring or cycloheteroalkyl ring can have 0-2 alkyl or 0-2 alkene substituents; R₅ can be hydrogen, hydroxy, lower alkyl, a lower alkene, a bond with an adjacent carbon, form a cycloalkyl ring with a ring atom of a ring formed by R₃ and R₄, wherein the cycloalkyl ring can have 0-2 double bonds, and the cycloalkyl ring can have 0-2 alkyl or 0-2 alkene substituents; each R₆ can separately be hydrogen, lower alkyl, lower alkene, or form a bond with an adjacent carbon; R₇ can be lower alkyl, lower alkene, or form a cycloalkyl ring with a R₅, R₈ can be lower alkyl, hydroxy, phosphate, diphosphate, or form a bond with an adjacent carbon; and R₉ can be hydrogen, lower alkyl, lower alkene, ═CH₂, hydroxy, phosphate, diphosphate, form a bond with an adjacent carbon, form a cycloalkyl ring with R₄, or form a cycloheteroalkyl ring with R₄, wherein each cycloalkyl ring or cycloheteroalkyl ring can have 0-2 double bonds, and each cycloalkyl ring or cycloheteroalkyl ring can have 0-2 alkyl or 0-2 alkene substituents.
 14. The method of claim 10, wherein the terpene is a compound with a skeleton selected from Sk1-Sk14:

or combination thereof.
 15. The method of claim 10, wherein the terpene is one or more of the following compounds:

wherein: 